JP2006266899A - Liquid chromatographic analysis method and apparatus - Google Patents

Abstract

Analysis accuracy of a liquid chromatograph analysis method and apparatus is improved.
By measuring the salt concentration in the washing liquid after washing the separation column, introducing the eluent into the column based on the salt concentration remaining in the separation column, and appropriately washing the separation column. This prevents a sample containing salt from being introduced into the analyzer. In addition to improving the analysis accuracy of the liquid chromatographic analysis method and apparatus, the separation column can be washed appropriately, so that the contamination of the separation column can be suppressed and the life can be extended.
[Selection] Figure 1

Description

  The present invention relates to a liquid chromatograph analysis method and apparatus used for biochemical analysis and the like.

  There is a liquid chromatograph device as an analyzer widely used for chemical analysis including various biochemical analyzes such as biological sample analysis and clinical sample analysis. In this liquid chromatograph apparatus, a measurement object in a sample is separated using a separation column. When analyzing a sample containing various mixtures other than the measurement object, the sample is once adsorbed on the column. Later, a step of cleaning impurities such as salts other than the measurement object is necessary.

  In this impurity cleaning step, after a sample is introduced into the column, a cleaning solution is introduced for a predetermined time to clean the impurities, an eluent is introduced into the column, and the eluted measurement object is placed in the analyzer. Install and analyze.

  Patent Document 1 describes a technique for exerting a high separation ability on a complicated mixed sample. The liquid chromatograph analyzer described in Patent Document 1 has two or more analysis units each having a mobile phase and an analysis column, and a plurality of concentration columns.

JP 2003-254955 A

  In the liquid chromatograph analyzer described in Patent Document 1, high separation ability can be exhibited.

  However, in the prior art, after a sample is introduced into a separation column, a solution for washing is introduced for a predetermined period of time. Therefore, regardless of whether or not impurities such as salt remaining in the separation column are sufficiently washed, there may be a case where a sample containing the salt is introduced into the analyzer using the separation column. The present inventor found the point.

  In addition, although the salt remaining in the separation column is sufficiently washed, there may be a case where the solution for washing is continued to flow, and there is a problem that the analysis time becomes long. Is the headline.

  An object of the present invention is to improve the analysis accuracy of a liquid chromatographic analysis method and apparatus.

  The present invention relates to measuring a salt concentration in a washing liquid after washing a separation column, and introducing an eluent into the column based on the salt concentration remaining in the separation column, thereby appropriately washing the separation column.

  ADVANTAGE OF THE INVENTION According to this invention, the situation where the sample containing a salt will be introduce | transduced into an analyzer can be prevented, and the analysis precision of a liquid chromatograph analysis can be improved.

The above and other novel features and costs of the present invention will be described below with reference to the accompanying drawings.
(First embodiment)
1 and 2 are schematic configuration diagrams of a liquid chromatograph apparatus according to a first embodiment of the present invention. FIG. 1 is a diagram illustrating a state during cleaning of the separation column 103, and FIG. 2 is a diagram illustrating a state during sample analysis.

  1 and 2, the liquid chromatograph apparatus includes a valve (flow channel switching means) 108 that changes the flow path, and a liquid feeding section 110 that introduces the sample into the valve 108 and performs desalting / washing of the sample. , A pump (liquid feeding means) 105 for sending the eluent 107 to the valve 108, an electric conductivity measuring device (salt concentration measuring means) 101, a column (sample adsorption / elution means) 103 for separating the sample, and a control device 111. And is connected to the analyzer 102.

  First, the liquid feeding unit 110 will be described. The liquid delivery unit 110 includes a pump 104 that introduces the solution 106 into the valve 108 and a sample introduction device 109 that introduces the sample into the solution 106.

  As the solution 106, a solution having a high ratio of water is used. This solution 106 is selected considering the ease of mixing with the sample solution. In the first embodiment, as an example, an aqueous 0.1% HCOOH solution can be used.

  The liquid feeding speed of the pump 104 is appropriately selected according to the type of packing material and the inner diameter of the separation column 103. Here, the liquid feeding speed of the pump 104 can be set to 1 ml / min, for example.

  The sample introduction device 109 is disposed on the discharge side of the pump 104 and on the inflow side of the separation column 103, and has a function of introducing a sample into the column 103. As the sample introduction device 109, an auto sampler capable of quantitatively and automatically continuously analyzing a plurality of samples is usually used. Further, as the sample introduction device 109, a manual injector capable of quantitatively introducing a sample can be used although continuous analysis cannot be automatically performed. When using a manual injector, the sample is quantified using a syringe quantification device.

  Liquid feeding is performed from the pump 104, and sample introduction is started from the sample introduction device 109. Then, the sample is fed to the valve 108, introduced into the column 103, and held. Thereafter, liquid feeding from the pump 104 to the valve 108 is continued, and the salt and dirt adsorbed on the column 103 are washed away.

Next, the pump 105 will be described.
The pump 105 sends the solution 107 to the column 103 via the valve 108 and elutes the sample from the column 103. In the first embodiment, a reverse phase column is used as the column 103. For the solution 107, 100% CH ₃ CN containing 0.1% HCOOH can be used.

  The electrical conductivity measuring device 101 is a device that measures the conductivity of ionic species in a solution. The electrical conductivity measuring apparatus 101 measures the electrical conductivity of the solution after the solution as the cleaning liquid sent from the pump 104 passes through the column 103. By measuring the electric conductivity of the solution after passing through the column 103, it is possible to determine whether or not the column 103 has been sufficiently cleaned.

  The analyzer 102 is an apparatus for analyzing each sample component eluted from the separation column 103, and is connected to the downstream side of the separation column 103 by a switching operation of the valve 108.

  In the first embodiment, a mass spectrometer capable of qualitative / quantitative analysis of a sample is applied to the analyzer 102. In the first embodiment, an electro ion spray ion trap mass spectrometer can be applied.

  The mass spectrometer referred to here is a quadrupole mass spectrometer, a time-of-flight mass spectrometer, or a Fourier transform ion cyclotron mass spectrometer. Further, ESI (electro ion spray) was used as an interface between the flow path and the mass spectrometer. As an interface, APCI (atmospheric pressure chemical ionization method) can also be used.

  In addition to the mass spectrometer, the analyzer may be a UV-VIS absorbance detector, a fluorescence detector, or an electrochemical detector.

  The column 103 is arranged in the flow path of the valve 108, and is arranged so as to change whether the solution is supplied from the pump 104 or the solution is supplied from 105 by switching the flow path of the valve 108. ing. The column 103 adsorbs the measurement target component in the sample, and then the gradient eluent is introduced, whereby the adsorbed measurement target component is eluted and separated from the column 103.

  In the first embodiment of the present invention, a reverse phase column used as the most general separation column can be used.

  In the column 103, various columns can be used. However, the solution 107 used when eluting each measurement target component from the column cannot affect the measurement accuracy of the analyzer 102. For example, when an ion exchange column is used for the column 103, a large amount of salt is introduced into the analyzer 102, so that the ion exchange column cannot be used for the column 103.

  The control device 111 controls the switching of the flow path of the valve 108, the liquid feeding of the pump 104 and the pump 105, the measurement operation of the sample introduction device 109, the measurement operation of the analysis device 102, and the electrical conductivity measurement device 101.

  FIG. 3 is a diagram illustrating a relationship between the control device 111 and its control target. As shown in FIG. 3, the control device 111 performs signal transmission / reception with the valve 108, the pump 104, the pump 105, the sample introduction device 109, the analysis device 102, and the electrical conductivity measurement device 101 via the interface 113. Then, analysis progress information, analysis results, the degree of cleaning of the separation column 103, the degree of contamination, and the like are displayed on the output device 112.

  Next, a procedure for controlling operations of the pumps 104 and 105, the valve 108, and the like by the control device 111 will be described. FIG. 4 is an operation control flowchart performed by the control device 111. In the first embodiment, the measurement was performed using a sample having a one-component composition and containing a salt.

  First, in step S 401, the solution 106 is sent to the valve 108 by the pump 104, and the sample is introduced into the column 103. Thereafter, the column 103 is washed with the washing liquid from the pump 104 in order to remove the components other than the measurement target component from the column 103 in step S <b> 402 through the channel as it is.

  At this time, whether or not the salt other than the target measurement target component adsorbed on the column 103 has been removed by measuring the ion concentration of the cleaning liquid discharged from the column 103 with the electrical conductivity measuring apparatus 101 in step 403. Is confirmed.

  In other words, the electrical conductivity of the cleaning liquid measured by the electrical conductivity measuring device 101 is transmitted to the control device 111, and the ion concentration of the cleaning liquid is calculated from the transmitted electrical conductivity, and the column 103 is cleaned (desalted). Status) is determined. The column 103 is washed until it is determined that the column 103 has been sufficiently washed (desalted).

  After confirming the desalting from the column 103, the control device 111 switches the flow path of the valve 108 from the state of FIG. 1 to the state of FIG. That is, the flow path to the column 103 is changed from the flow path that becomes the pump 104 → the column 103 → the electric conductivity measuring apparatus 101 to the flow path that becomes the pump 105 → the column 103 → the analysis apparatus 102.

  Then, the eluent is sent from the solution 107 to the column 105, so that the sample components are eluted and introduced into the analyzer 102, and the sample is measured (steps S404 and S405).

  This series of measurements can be performed at least once under the control of the control device 111.

  As described above, after measurement is performed by the electrical conductivity measuring apparatus 101 and the desalting of the column 103 is completely performed, the flow path is switched, and the solution is fed from the pump 105 to the column 103, and the analyzing apparatus 102 The component to be measured is supplied to.

  Therefore, even when the salt concentration in the sample is high, the solution containing the measurement object is introduced into the analyzer 102 from the column 103 in a state where the desalting process has been sufficiently performed.

  In the first embodiment of the present invention, a mass spectrometer is used as the analyzer. In general, if the desalting of the column 103 is not performed completely, the salt contained in the sample component is mixed in the mass spectrometer, and the performance is affected by the precipitation of the salt.

  In that respect, in the first embodiment, since it can be confirmed that the desalting has been completely performed from the column 103, it is possible to prevent the situation that the salt contained in the sample component is mixed into the mass spectrometer and improve the analysis accuracy. In addition, contamination of the mass spectrometer can be prevented.

  As described above, according to the first embodiment of the present invention, a liquid chromatograph analysis method and apparatus capable of improving analysis accuracy can be realized.

  In addition, since the separation column can be washed appropriately, contamination of the separation column can be suppressed and the life can be extended.

  In the prior art, the desalting operation is performed for a certain period of time regardless of whether or not the desalting is actually completed. For this reason, even when the desalting is completed, the washing solution is passed through the column until a predetermined time elapses, resulting in excessive use of the time and the washing solution.

  On the other hand, in the first embodiment of the present invention, when the desalting of the column 103 is completely finished regardless of the passage of time, the desalting treatment of the column is finished and the process proceeds to the next step. Therefore, desalting is performed only for an appropriate time. That is, it is possible to avoid the desalting treatment time and the excessive use of the cleaning solution. As a result, the analysis time can be shortened and the analysis throughput can be improved.

  In addition, since the salt introduced into the column is surely washed, the deterioration of the column can be prevented and the life can be extended. Furthermore, it is possible to prevent a decrease in resolution due to the column, and the analysis accuracy is improved.

  Further, in the prior art, it is necessary to appropriately determine a program in consideration of a cleaning (desalting) time. In that respect, in the first embodiment of the present invention, it is possible to automatically determine that the cleaning (desalting) step has ended. That is, it is possible to omit troublesome parameter determination work at the start of measurement, and the convenience of the apparatus is improved.

(Second Embodiment)
Next, a second embodiment of the present invention will be described.
Currently, as a result of various genome projects at the national level, there are a wide variety of species whose genome sequence has been clarified, including humans, plants, and microorganisms. Based on this genomic information, proteomics is an active area that grasps the expression information of the genome and comprehensively analyzes the dynamics of proteins produced in specific organs, tissues, and cells. Proteomics focuses on analyzing the dynamics of proteins in organs, tissues, and cells by analyzing them using protein profiling techniques.

  In this profiling technique, analysis is generally performed by a combination of two-dimensional electrophoresis and a mass spectrometer.

  However, due to recent technological innovations in the proteome field, the method has changed to two-dimensional electrophoresis, and a method of separating sample components using a high-speed chromatograph combining different types of columns and introducing them into a mass spectrometer is being used.

  The second embodiment of the present invention is an embodiment relating to a method and an apparatus for carrying out separation easily and efficiently with high accuracy for multidimensional high-speed chromatography used for profiling proteins in the field of proteomics.

  5 and 6 are schematic configuration diagrams of the multidimensional high-speed chromatograph apparatus according to the second embodiment of the present invention, and FIG. 5 is a diagram showing states during sample concentration and washing in the concentration column 504. FIG. 6 is a diagram showing a state during analysis of the sample.

The second embodiment of the present invention is composed of five components.
The first component is a pre-stage liquid feeding means (pump) 512 that introduces the gradient eluent into the liquid feeding flow path at a micro flow level (μl / min) quantification, and a sample introduction that introduces the sample into the liquid feeding flow path. A first-dimensional separation unit 523 including a device 506 and a first-dimensional separation column 503 that separates a sample into components.

  The second component includes a column 504 for holding each separated component separated by the first-dimensional separation unit 523, and a solution is sent from the pump 503 in the first component, and each separated component is washed (desalted). ) And a desalting unit 524 including an electrical conductivity measuring machine 501 for measuring the cleaning liquid.

  The third component is for each component separated by the post-stage liquid feeding means 518 for introducing the gradient eluent into the liquid feeding flow path at a nanoflow level (nl / min) and the first-dimensional separation unit 523. Further, a second-dimensional separation unit 525 including a second-dimensional separation column 505 that performs separation.

  The fourth component is a control device 522 that controls the first to third components and the analyzer unit 526.

  Each component separated by the second dimensional separation unit 525, which is the third component, is analyzed by the analyzer unit 526.

  First, the configuration of the first-dimensional separation unit 523 will be described.

  As the sample introduction device 506, an auto sampler capable of quantitatively and automatically continuously analyzing a plurality of samples was used. It is also possible to introduce a sample using a manual injector and a syringe.

  The pump 512 mixes the two solutions 508 and 509 at a flow rate of micro flow level (μl / min) by determining the composition ratio of the solutions 508 and 509 by a low pressure gradient method, that is, by turning on / off the solenoid valves 510 and 511. It is possible to perform liquid feeding. The composition ratio of the solutions 508 and 509 can be automatically controlled by the control device 522.

  The solution sent from the pump 512 is mixed by the mixer 513 and then sent to the first-dimensional separation column 503. The composition of the solutions 508 and 509 is determined by the characteristics of the first-dimensional separation column 503.

  In the second embodiment, since an ion exchange column is used for the first-dimensional separation column 503, gradient eluents having different ionic strengths can be created and used. This pump 512 can also be applied to the case of using a low pressure gradient method in which a plurality of three or four types of solutions are mixed.

The solution 508 can be a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH, and the solution 509 can be a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH and 1 mol / l HCOONH ₄ .

  The first-dimensional separation column 503 is an ion exchange column, and various ion exchange resins are appropriately used as a stationary phase depending on the properties of the eluent and the sample in terms of the properties of the sample.

  The sample introduced from the sample introduction device 506 is held in the ion exchange column 503 based on the ionicity of each component in the sample. Thereafter, the gradient eluent having different ionic strengths created by the pump 512 is introduced into the ion exchange column 503, whereby the sample is separated by the difference in affinity to the ion exchange resin packed in the ion exchange column 503.

  In the second embodiment, as the first-dimensional separation column 503, for example, a column having an inner diameter of 300 μm and a length of 150 mm can be used.

Next, the desalting unit 524 will be described.
The desalting unit 524 has a function of desalting each sample component separated by the ion exchange column that is the first-dimensional separation column 503 of the first-dimensional separation unit 523.

  Each sample component separated by the first-dimensional separation unit 523 is sent to the valve 507 and introduced into the concentration column 504 to concentrate the sample. The concentration column 504 is a column in which various kinds of fillers are used as a stationary phase in terms of sample properties and the like, and are filled with a lower volume of the filler than the separation column.

  Here, the pump 512 of the first-dimensional separation unit 523 described above sends the solution 508 and the solution 509 at a flow rate of microflow level (μl / min). The pump 512 also has a function of washing (desalting) each sample component concentrated in the concentration column 504 of the desalting unit 524 in addition to the function of feeding the sample.

As the cleaning liquid for cleaning the concentration column 504, a liquid having the same composition as the initial eluent used in the second-dimensional separation unit 525 is used. In general, a solution containing a low concentration organic acid is often used. In this second embodiment, the solution 508 can be a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH.

  The electrical conductivity measuring device 501 of the desalting unit 524 is a device that measures the conductivity of ionic species in the solution. The electrical conductivity measuring device 501 measures the conductivity of the solution sent from the pump 512 as a cleaning liquid after passing through the concentration column 504. The same effect can be obtained by using a conductivity detector, an ultraviolet absorption detector, or an electrochemical detector in the electrical conductivity measuring apparatus 501.

Next, the second-dimensional separation unit 525 will be described.
The pump 518 mixes the two solutions 514 and 515 at a flow rate of nanoflow level (nl / min) with a low pressure gradient method, that is, with the solenoid valves 516 and 517 turned on / off, with their composition ratios determined and mixed. I do. Note that these composition ratios are controlled by the control device 522.

  The solution sent from the pump 518 is mixed by the mixer 519 and then sent to the valve 507. The composition of the solutions 514 and 515 is determined by the characteristics of the second dimension separation column 505. In the second embodiment, since a reversed-phase column is used as the second-dimensional separation column 505, gradient eluents having different polarities can be created and used.

The pump 518 may use a low pressure gradient method in which a plurality of three or four types of solutions are mixed. The solution 514 can be a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH, and the solution 515 can be a 98% CH ₃ CN aqueous solution containing 0.1% HCOOH.

  The second-dimensional separation column 505 is a reversed-phase column, and various stationary phases are appropriately selected depending on the properties of the eluent and the sample in terms of the properties of the sample.

  By switching the valve 507 from the state shown in FIG. 5 to the state shown in FIG. 6, the pump 518 is switched to a flow path passing through the concentration column 504 and the second-dimensional separation column 505.

  Then, the gradient solution from the pump 518 is sequentially sent to the concentration column 504 where each sample component is concentrated, the sample is eluted, and each sample component reaches the second-dimensional separation column 505 sequentially.

Next, the analyzer unit 526 will be described.
As the analyzer 502, a mass spectrometer is usually used. An apparatus combining this multi-dimensional high-speed chromatograph and a mass spectrometer is widely used as a means for proteomic analysis, that is, for analyzing proteins that interact in a complex manner in vivo and in cells.

  When measuring a normal sample, it is separated by one kind of column and detected by an analyzer, but in this second embodiment, the main purpose is to analyze and detect a mixed sample made of various proteins. The sample is separated using two types of separation columns 503 and 505.

  In many cases, a protein mixture sample as an analysis sample is digested with an enzyme and previously decomposed into peptide fragments. For this reason, it is introduced into the ion exchange column 503 by the first-dimensional separation unit 523, and is separated by the ionic strength, that is, the affinity to the ion exchange resin. Then, concentration is performed in the concentration column 504 of the desalting unit 524, and cleaning (desalting) is performed by feeding the cleaning liquid from the pump 512. Then, it introduce | transduces into the reverse phase column 505 of the 2nd dimension separation part 525, and also isolate | separates.

  Thus, the separated sample components are introduced into the mass spectrometer unit 526, and the amino acid sequence of the peptide fragment is identified. The identified amino acid sequence information is collated with an amino acid sequence information search database to identify a protein.

  An electro ion spray ion trap mass spectrometer was used as the mass spectrometer. As the mass spectrometer used here, a quadrupole mass spectrometer, a time-of-flight mass spectrometer, and a Fourier transform ion cyclotron mass spectrometer can be applied.

  In addition to the mass spectrometer, the analyzer can be a UV-VIS absorbance detector, a fluorescence detector, or an electrochemical detector.

Next, the control device 522 will be described.
The control device 522 controls the composition ratio of the solution and the liquid feeding operation in the pumps 512 and 518, the introduction of the sample from the sample introduction unit (autosampler) 506, the switching of the flow path of the valve 507, and the measurement operation of the electrical conductivity measuring device 501. The measurement operation of the analyzer 502 is controlled. FIG. 7 is a diagram illustrating a relationship between the control device 522 and the control target. As illustrated in FIG. 7, the control device 522 performs signal transmission / reception with the valve 507, the pump 512, the pump 518, the sample introduction device 506, the analysis device 502, and the electrical conductivity measurement device 501 via the interface 527. Then, analysis progress information, analysis results, cleaning degree of the concentration column 504, contamination degree, and the like are displayed on the output device 528.

  Next, the control operation of the control device 522 will be described with reference to the operation flowchart shown in FIG.

  First, in step S <b> 801, a sample is introduced from the sample introduction device 506 into the first-dimensional separation column 503 by liquid feeding from the pump 512. The composition from the pump 512 is controlled by the controller 522 and is introduced into the first-dimensional separation column 503 for a predetermined time.

  Next, in step S 802, the target sample is eluted from the separation column 503 using the salt concentration gradient from the pump 512, and concentrated in the concentration column 504. Thereafter, the desalination treatment of the concentration column 504 is started by the eluent from the pump 512, and the salt concentration of the solution discharged from the concentration column 504 is measured by the electric conductivity measuring device 501, and the concentration column 504 is dehydrated. The process is performed until the salt is completed (steps S803 and S804).

  In steps S801 to S804, the introduction of the sample into the concentration column 504 and the desalting treatment of the concentration column 504 are performed in accordance with a predetermined salt so that the salt concentration gradually increases as shown in FIG. The process is repeated alternately until the concentration is reached. That is, from the time point 0 to t1 in FIG. 9 (a), after the salt concentration is increased in an inclined manner, the desalting treatment of the column 504 is started, and the salt concentration of the solution discharged from the column 504 becomes zero. The desalting treatment is performed until time t2 ((b) of FIG. 9). Then, after completion of the desalting treatment, the salt concentration is increased from time t2 to t3 so that the next salt concentration is obtained, and then the desalting treatment of the column 504 is started and the salt of the solution discharged from the column 504 is started. The desalting process is performed until time t4 when the concentration becomes zero. Thereafter, similarly, the salt concentration is increased in a gradient manner until a predetermined concentration is reached, and desalting is performed.

  Subsequently, the flow path of the valve 507 is switched, and the flow path for supplying the solution to the concentration column 504 is switched from the pump 512 to the pump 518. That is, the flow path shown in FIG. 5 is switched to the flow path shown in FIG. 6 (step S805).

  Then, using the eluent from the pump 518, the target component among the sample components from the concentration column 504 is separated by the second-dimensional separation column 505 (step S806). The target component separated by the second-dimensional separation column 505 is introduced into the analyzer 502 by liquid feeding from the pump 518, and the sample component is measured (step S807).

  When the measurement by the analyzer 502 is completed, the flow path of the valve 507 is switched from the state shown in FIG. 6 to the state shown in FIG. 5 (step S808), and the process returns to step S8012.

  As described above, according to the second embodiment of the present invention, the measurement is performed by the electrical conductivity measuring device 501, and after the desalination of the concentration column 504 is completely performed, the controller 512 outputs the pump 512. Is switched to liquid feeding to the pump 518.

  Therefore, also in the second embodiment, the same effect as that of the first embodiment can be obtained.

(Third embodiment)
The third embodiment of the present invention is an example in the case where the present invention is applied to a multidimensional high-speed chromatograph apparatus, similarly to the second embodiment described above. The difference between the third embodiment and the second embodiment is that the method of introducing the eluent into the first-dimensional separation column is different. That is, the configuration of the first-dimensional separation unit is different from the operation of the control device. The other points are the same as in the second embodiment, and thus the description thereof is omitted.

FIG. 10 is a schematic configuration diagram of the second embodiment of the present invention. In FIG. 10, the pump 1009 of the first-dimensional separation unit 1019 sends the solution from the solution 1008 at a flow rate of microflow level (μl / min), and introduces the sample into the first-dimensional separation column 1003 via the sample introduction device 1006. To do. Here, as the solution 1008, in this third embodiment, a 2% CH ₃ CN aqueous solution containing 0.1% HCOOH can be used.

  The sample introduction device 1006 is disposed on the downstream side (discharge side) of the pump 1009 and on the upstream side of the first-dimensional separation column 1003. In general, an autosampler capable of quantitatively and automatically continuously analyzing a plurality of samples is used as the sample introduction device 1006. The sample introduction device 1006 has a function of introducing a sample into the first-dimensional separation column 1003 and a function of introducing an eluent into the first-dimensional separation column 1003. In the third embodiment, an ion exchange column can be used as the first-dimensional separation column 1003.

The sample introduction device 1006 has a sample in the vial 1, a vial 2 with 2% CH ₃ CN, 0.1% HCOOH, 10 mM HCOONH ₄ aqueous solution, a vial 3 with 2% CH ₃ CN, 0.1% HCOOH, 20 mM HCOONH ₄ aqueous solution, 2% CH ₃ CN, 0.1% HCOOH, 50 mM HCOONH ₄ aqueous solution in vial 4, 2% CH ₃ CN, 0.1% HCOOH, 100 mM HCOONH ₄ aqueous solution in vial 5, 2% in vial 6 CH ₃ CN, 0.1% HCOOH, 200 mM HCOONH ₄ aqueous solution, and vial 7 can be 2% CH ₃ CN, 0.1% HCOOH, 500 mM HCOONH ₄ aqueous solution.

  An operation of introducing a sample or a gradient eluent by the sample introduction device 1006 will be described.

  First, a sample is introduced from the vial 1 into the first-dimensional separation column 1003 for a predetermined time programmed by the pump 1009. Next, several types of samples and solutions having different ionic strengths are arranged on the rack of the sample introduction device 1006, and sequentially introduced into the first-dimensional separation column 1003 under the control of the control device 1018. By this operation, the eluents having different ionic strengths are introduced into the first-dimensional separation column 1003 on which the sample is adsorbed.

  That is, the sample from the vial 1 is separated by the first-dimensional separation column 1003 and introduced into the desalting unit 524. After the sample from the vial 1 is introduced, the eluent is sent from the vial 2, separated into each sample component by the first-dimensional separation column 1003, and then concentrated by the concentration column 504.

  In the meantime, liquid feeding by the pump 1009 is performed, and the concentration column 504 is washed (desalted). That is, as shown in FIG. 11, the concentration column 504 is washed during the period from time t1 to t2, from time t3 to t4, from t5 to t6,.

  During this washing (desalting) step, the electrical conductivity measuring device 501 measures the ion concentration of the washing liquid discharged from the concentration column 504. At this time, if the ion concentration measured by the electrical conductivity measuring device 1001 is lower than a preset value, it is determined that the desalting of the concentration column 1004 has been completed. When the desalting of the condensed column 1004 is completed, the flow path of the valve 507 is switched by the control device 1018 to switch from the liquid feeding from the pump 1009 to the concentration column 504 to the liquid feeding from the pump 518 to the concentration column 504. . At this point, the desalting process is complete.

  In synchronization with the switching of the flow path of the valve 507, the eluent sent from the pump 518 is sent to the second-dimensional separation column 505 via the concentration column 504, and the second-dimensional separation starts. The sample thus separated is introduced into the analyzer 502 and measurement is started.

  When the measurement by the analyzer 502 is completed, the flow path of the valve 507 is switched to the state shown in FIG. 10, and the eluent of the vial 3 is introduced into the first-dimensional separation column 1003. Thereafter, the sample separation is performed in the same manner as described above. After concentration, separation, measurement is performed.

  The series of operation control described above are all executed by the control device 1018, and the sample can be measured automatically and continuously.

  In the third embodiment of the present invention, the same effect as that of the first embodiment can be obtained.

  Here, in the first to third embodiments described above, the ion concentration of the cleaning liquid discharged from the separation column or the concentration column in the cleaning step after the sample is introduced into the separation column or the concentration column is determined based on the electric conductivity. Measured with a measuring device to confirm the completion of desalting.

  In the present invention, the washing solution is supplied to the separation column or concentration column not only in the washing step but also before the sample is introduced into the separation column or concentration column, and the desalted state is confirmed by an electrical conductivity measuring device. You can also.

  Further, the ion concentration of the cleaning liquid itself can be detected by an electrical conductivity measuring device, an ultraviolet absorption detector, an electrochemical detector, or the like. As a result, it is possible to detect that the cleaning liquid is contaminated, and it is possible to replace the cleaning liquid with an uncontaminated one before analyzing the sample. This is because if the cleaning liquid is contaminated, the analysis accuracy of the sample is lowered.

It is a schematic block diagram of the liquid chromatograph apparatus which is the 1st Embodiment of this invention. It is operation | movement explanatory drawing of the liquid chromatograph apparatus which is the 1st Embodiment of this invention. It is a figure which shows the relationship between the control apparatus in the 1st Embodiment of this invention, and its control object. It is an operation | movement flowchart in the 1st Embodiment of this invention. It is a schematic block diagram of the liquid chromatograph apparatus which is the 2nd Embodiment of this invention. It is operation | movement explanatory drawing of the liquid chromatograph apparatus which is the 2nd Embodiment of this invention. It is a figure which shows the relationship between the control apparatus in the 2nd Embodiment of this invention, and its control object. It is an operation | movement flowchart in the 2nd Embodiment of this invention. It is a figure which shows the relationship between the salt concentration of the liquid feeding from the pump in 2nd Embodiment of this invention, and the salt concentration measured by an electrical conductivity measuring apparatus. It is a schematic block diagram of the liquid chromatograph apparatus which is the 3rd Embodiment of this invention. It is a figure which shows the relationship between the salt concentration of the liquid feeding from the pump in 3rd Embodiment of this invention, and the salt concentration measured by an electrical conductivity measuring apparatus.

Explanation of symbols

101, 501 Electrical conductivity measuring device 102, 502 Analysis device 103, Separation column 104, 105 Pump 108, 507 Valve 109, 506 Sample introduction device 110 Liquid feeding part 111 Control device 503 First dimension separation column 504 Concentration column 505 Two dimension Eye separation column 510, 511 Electromagnetic valve 512, 518 Pump 513 Mixer 516, 517 Electromagnetic valve 519 Electromagnetic valve 522 Control device 523 First-dimensional separation unit 524 Desalination unit 525 Second-dimensional separation unit 526 Analytical device unit 1003 First-dimensional separation Column 1006 Sample introduction device 1009 Pump

Claims (18)

In the liquid chromatographic analysis method,
Introduce the sample together with the salt-containing solution to the first column that can selectively adsorb and elute the sample to adsorb the measurement object,
The solution is introduced into the first column and washed, the salt concentration of the solution discharged from the first column is measured, and whether the washing is continued or stopped based on the measured salt concentration. Judgment
After determining that the washing to the first column is stopped, the solution is introduced into the first column, and the measurement object adsorbed on the first column is eluted from the first column. A characteristic liquid chromatographic analysis method.   2. The liquid chromatographic analysis method according to claim 1, wherein the salt concentration is measured by measuring the electrical conductivity of the solution discharged from the first column.   3. The liquid chromatographic analysis method according to claim 2, wherein the salt concentration of the solution containing the salt is adjusted and introduced into the second column together with the sample to adsorb the sample measurement object onto the second column. Thereafter, the solution is introduced into the second column, and the adsorbed measurement object is eluted and introduced into the first column, and the salt concentration of the solution discharged from the first column is measured. Then, based on the measured salt concentration, it is determined whether to continue or stop the introduction of the solution into the first column, and after determining that the introduction of the solution into the first column is stopped, The solution is introduced into the first column, the measurement object adsorbed on the first column is eluted from the first column, is introduced into the third column, and the measurement object is adsorbed. Introducing the solution into the third column, Liquid chromatography analysis wherein the eluting the measuring object adsorbed to the column from the third column.   4. The liquid chromatographic analysis method according to claim 3, wherein the salt concentration of the solution introduced into the second column is increased in an inclined manner with time.   4. The liquid chromatographic analysis method according to claim 3, wherein the salt concentration of the solution introduced into the second column is increased stepwise with time.   2. The liquid chromatographic analysis method according to claim 1, wherein a cleaning solution is introduced into the first column and washed before introduction of the sample into the first column, and the sample is discharged from the first column. And measuring the salt concentration of the solution and determining the degree of salt contamination of the first column based on the measured salt concentration. In a liquid chromatograph analyzer,
A first column capable of selectively adsorbing and eluting a sample;
A first pump for feeding a solution containing salt to the first column;
A sample introduction device for introducing a sample into the solution fed from the first pump;
A salt concentration measuring device for measuring the salt concentration of the cleaning solution introduced into and discharged from the first column by the first pump;
A second pump for introducing a sample elution solution into the first column and eluting the sample components adsorbed on the first column;
A valve having a flow path for introducing the solution from the first pump to the first column and a flow path for introducing the solution from the second pump to the first column, and one of the flow paths can be selected;
Based on the salt concentration measured by the salt concentration measuring device, it is determined whether to continue or stop the introduction of the cleaning solution, and the controller controls the operations of the first pump, the second pump, and the valve. When,
A liquid chromatograph analyzer comprising:   8. The liquid chromatograph analyzer according to claim 7, wherein the salt concentration measuring device is an electric conductivity measuring device and measures the salt concentration by measuring the electric conductivity of the solution discharged from the first column. A liquid chromatograph analyzer characterized by that. The liquid chromatograph analyzer according to claim 8,
A second column capable of selectively adsorbing and eluting the sample sent from the first pump;
A third column capable of selectively adsorbing and eluting the sample eluted from the first column;
The control unit adjusts the salt concentration of the solution containing the salt, introduces the sample together with the sample into the second column, and adsorbs the sample measurement object on the second column, and then The solution is introduced into the second column, the adsorbed measurement object is eluted and introduced into the first column, and the salt concentration of the solution discharged from the first column is measured and measured. Based on the measured salt concentration, it is determined whether to continue or stop the introduction of the solution into the first column, and after determining that the introduction of the solution into the first column is stopped, the first column The measurement object adsorbed on the first column is eluted from the first column and introduced into the third column to adsorb the measurement object, and then the third column is introduced. The solution was introduced into the column and adsorbed on the third column. Liquid chromatographic analysis apparatus characterized by eluting the object from the third column.   10. The liquid chromatograph analyzer according to claim 9, wherein the control unit increases the salt concentration of the solution introduced into the second column in an inclined manner with time. .   10. The liquid chromatograph analyzer according to claim 9, wherein the controller increases the salt concentration of the solution introduced into the second column stepwise with time. .   8. The liquid chromatograph analyzer according to claim 7, wherein the control unit introduces a cleaning solution into the first column and cleans the sample before introducing the sample into the first column. A liquid chromatograph analyzer characterized by measuring the salt concentration of the solution discharged from the column and determining the degree of salt contamination of the first column based on the measured salt concentration. In a liquid chromatograph analyzer,
A first sample adsorption / elution means capable of selectively adsorbing / eluting a sample;
First liquid feeding means for feeding a solution containing salt to the first sample adsorption / elution means;
Sample introduction means for introducing a sample into the solution fed from the first liquid feeding means;
A salt concentration measuring means for measuring the salt concentration of the cleaning solution introduced into and discharged from the first sample adsorption / elution means by the first liquid feeding means;
A second liquid feeding means for introducing a sample elution solution into the first sample adsorption / elution means and eluting the sample components adsorbed by the first sample adsorption / elution means;
A flow path for introducing the solution from the first liquid feeding means to the first sample adsorption / elution means and a flow path for introducing the solution from the second liquid feeding means to the first sample adsorption / elution means, A flow path switching means capable of switching and selecting any flow path;
Based on the salt concentration measured by the salt concentration measuring device, it is determined whether to continue or stop the introduction of the cleaning solution, and the first liquid feeding means, the second liquid feeding means, and the flow path switching means Control means for controlling the operation;
A liquid chromatograph analyzer comprising:   14. The liquid chromatograph analyzer according to claim 13, wherein the salt concentration measuring means is an electric conductivity measuring device, and the salt concentration is measured by measuring the electric conductivity of the solution discharged from the first sample adsorbing / eluting means. A liquid chromatograph analyzer characterized by measuring a concentration. The liquid chromatograph analyzer according to claim 14,
A second sample adsorption / elution means capable of selectively adsorbing / eluting the sample fed from the first liquid feeding means;
A third sample adsorption / elution means capable of selectively adsorbing / eluting the sample eluted from the first sample adsorption / elution means;
And the control means adjusts the salt concentration of the solution containing the salt, introduces the sample together with the sample to the second sample adsorption / elution means, and supplies the sample measurement object to the second sample adsorption / elution means. Then, the solution is introduced into the second sample adsorption / elution means, the adsorbed measurement object is eluted and introduced into the first sample adsorption / elution means, and the first sample adsorption / elution means is introduced. Measure the salt concentration of the solution discharged from the sample adsorption / elution means, and determine whether to continue or stop the introduction of the solution into the first sample adsorption / elution means based on the measured salt concentration Then, after it is determined that the introduction of the solution to the first sample adsorption / elution means is stopped, the solution is introduced to the first sample adsorption / elution means and adsorbed to the first sample adsorption / elution means. The measured object to be measured is eluted from the first sample adsorption / elution means. It is introduced into the third sample adsorption / elution means to adsorb the measurement object, and then the solution is introduced into the third sample adsorption / elution means and adsorbed to the third sample adsorption / elution means. A liquid chromatograph analyzer characterized in that a measurement object is eluted from the third sample adsorption / elution means.   16. The liquid chromatograph analyzing apparatus according to claim 15, wherein the control means increases the salt concentration of the solution introduced into the second sample adsorption / elution means in an inclined manner with the passage of time. Chromatographic analyzer.   16. The liquid chromatograph analysis apparatus according to claim 15, wherein the control means increases the salt concentration of the solution introduced into the second sample adsorption / elution means in a stepped manner with time. Chromatographic analyzer.   14. The liquid chromatograph analyzing apparatus according to claim 13, wherein the control means supplies a cleaning solution to the first sample adsorption / elution means before introducing the sample into the first sample adsorption / elution means. The solution is introduced and washed, and the salt concentration of the solution discharged from the first sample adsorption / elution means is measured, and the degree of salt contamination of the first sample adsorption / elution means is determined based on the measured salt concentration. A liquid chromatograph analyzer characterized by that.

Images