JP2014145675A - Analyzer, and method of obtaining chromatogram of liquid sample using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase the speed of analysis by adjusting the flow rate so as to increase the column pressure to the upper limit in a liquid chromatograph using a postcolumn derivatization method.SOLUTION: In order to suppress the detection base line variation caused in accordance with a gradient elution program of an eluant, the flow rate of a first pump for feeding the eluant and the flow rate of a second pump for feeding a derivatization reagent are varied in interlock with the program. At this time, the flow rates of the first/second pumps are varied in accordance with a preset program so as to increase the column pressure to the upper limit, the speed of the analysis is increased, and a reaction environment condition is established at a high reproducibility for each analysis injection cycle.

Description

  The present invention relates to an analyzer and a method for acquiring a chromatogram of a liquid sample using the same, for example, a technique for acquiring a liquid chromatogram equipped with a reaction system using a post-column derivatization method. .

  Conventionally, there is an amino acid analyzer as a typical apparatus for a liquid chromatograph using a post-column derivatization method. For example, as described in Patent Document 1, according to a conventional amino acid detection method, a ninhydrin reagent is added to a buffer solution (eluent) containing an amino acid component separated by a separation column fed by a buffer pump. The mixed solution is reacted by passing through a heated reaction coil or reaction column, and continuously detected by a visible detector. This reaction coil normally uses a thin, long Teflon (registered trademark) tube of 0.25 mm ID × 7-20 m in order to obtain a constant reaction time while flowing the reaction mixture. In the case of a reaction column, the reaction time is obtained using a columnar cylinder packed with 10 to 100 μm inert beads.

JP-A-4-194750

  Regardless of the stepwise elution method or the linear gradient elution method, the liquidity and flow rate of the eluent sent to the separation column are often changed to improve chromatographic separation. Liquidity is characterized by pH, salt concentration, organic solvent concentration, and other modifier concentrations. In the case of the post-column derivatization method, when the liquidity or flow rate of the eluent changes, the detection signal value is affected and the baseline often fluctuates. This is because changes in the reaction environment and reaction time of the eluent and the derivatization reagent affect the absorbance and fluorescence intensity of the mixed solution, thereby changing the absorbance detection and fluorescence detection signal values. .

  In this respect, in the sample detection by the conventional liquid chromatograph, since the flow rate of each eluent is kept constant, the pressure applied to the separation column varies due to the difference in the viscosity of each eluent. On the other hand, a pressure resistance standard (maximum pressure resistance) is set for the separation column, and there is a risk that the separation column will break down if it is continuously used at a pressure exceeding the pressure resistance standard. For this reason, the conventional detection method supports eluents of various viscosities, so that even if the pressure applied to the separation column fluctuates, the pressure is much lower than the pressure resistance standard so as not to exceed the pressure resistance standard. The flow rate is controlled so as to be suppressed.

  However, detection by such constant flow rate control requires a long time for analysis, and there is a problem that the efficiency for sample analysis deteriorates.

  The present invention has been made in view of such a situation, and shortens the analysis time in a liquid chromatograph using the post-column derivatization method and contributes to improvement in analysis efficiency.

(I) In order to solve the above-described problem, in the present invention, in order to suppress fluctuations in the detection baseline that change with the gradient elution program of the eluent, the flow rate of the second pump that sends the derivatization reagent is set in the program. It changes more or less in conjunction with. Here, the flow rate of the second pump is changed by a preset program, and the reaction environment condition is established with high reproducibility for each analysis injection cycle. For example, when the absorbance of the reaction mixture is slightly increased, the flow rate of the second pump is slightly reduced. In the opposite case, the flow rate is slightly increased.

  In addition, the second pump may mix and feed the reaction reagent and the buffer that creates the optimum reaction conditions. In this case, in addition to the method of changing the second pump, for example, when the absorbance of the reaction mixture is slightly increased, the mixing ratio of the buffer solution can be increased. Conversely, when the detection signal such as absorbance changes slightly smaller, the mixing ratio of the reaction reagent is increased.

(Ii) That is, the analyzer according to the present invention includes a first pump for feeding an eluent, a second pump for feeding a derivatization reagent, and a first flow extending from the first pump. A path, a second flow path extending from the second pump, and a sample introduction section for introducing a sample to be analyzed provided in the first flow path after the first pump; In the first channel, a separation mechanism for separating each component of the sample provided at the subsequent stage of the sample introduction unit, and provided at the subsequent stage of the separation mechanism, the second channel is connected to the first channel, and the sample And a detector for detecting the reaction between the sample and the derivatizing reagent and outputting a detection signal, and controlling each component of the analyzer, and a chromatogram based on the detection signal. And a data processing device for generating and outputting the data. Here, the data processing apparatus introduces the flow rate of the eluent into the first flow path over time, and follows a given analysis program for keeping the pressure of the liquid applied to the separation mechanism within a predetermined range. Control the analyzer. Specifically, the data processing apparatus controls the flow rate (variation amount) of the eluent by operating the liquid feeding operation of the first pump based on the analysis program, and the pressure applied to the separation mechanism from the detection signal. Acquire a pressure profile showing the change over time and a chromatogram of the mixture.

(Iii) Further features related to the present invention will become apparent from the description of the present specification and the accompanying drawings. The embodiments of the present invention can be achieved and realized by elements and combinations of various elements and the following detailed description and appended claims.

  It should be understood that the description herein is merely exemplary and is not intended to limit the scope of the claims or the application of the invention in any way.

  According to the present invention, the analysis time in a liquid chromatograph using a post-column derivatization method is shortened, which contributes to improvement in analysis efficiency.

It is a figure which shows schematic structure of the analyzer by embodiment of this invention. It is a flowchart for demonstrating the whole operation | movement of embodiment of this invention. It is a figure which shows the example of a gradient elution program. It is a figure which shows the example of the chromatogram obtained by execution of the program of FIG. It is a figure which shows the example of the pressure profile obtained by execution of the program of FIG. It is a figure which shows the example of the improved gradient elution program (improvement analysis program). It is a figure which shows the example of the chromatogram acquired according to the improvement analysis program. It is a figure which shows the example of the pressure profile acquired according to the improvement analysis program.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be denoted by the same numbers. The attached drawings show specific embodiments and implementation examples based on the principle of the present invention, but these are for understanding the present invention and are not intended to limit the present invention. Not used.

  This embodiment has been described in sufficient detail for those skilled in the art to practice the present invention, but other implementations and configurations are possible without departing from the scope and spirit of the technical idea of the present invention. It is necessary to understand that the configuration and structure can be changed and various elements can be replaced. Therefore, the following description should not be interpreted as being limited to this.

  Furthermore, as will be described later, the embodiment of the present invention may be implemented by software running on a general-purpose computer, or may be implemented by dedicated hardware or a combination of software and hardware.

  In the following, each process in the embodiment of the present invention will be described with “data processing device (computer or processor)” as the subject (operation subject), but the description is based on the processing program that defines the operation according to FIG. It is good. Further, the processing disclosed with the program as the subject may be processing performed by a computer such as a management server or an information processing apparatus. Part or all of the program may be realized by dedicated hardware, or may be modularized. Various programs may be installed in each computer by a program distribution server or a storage medium.

<Configuration of amino acid analyzer>
FIG. 1 is a diagram showing a schematic configuration of an amino acid analyzer according to an embodiment of the present invention. In the present embodiment, the operation of the amino acid analyzer is described as an example, but other analyzers may be used.

  According to FIG. 1, the amino acid analyzer 100 includes an eluent liquid feeding mechanism 11, a sample introduction unit 3, a separation mechanism 12, a reaction liquid feeding mechanism 13, a T-joint 7, a reaction apparatus 8, The visible detector 9, the flow paths 14 and 15 connecting them, and the data processing device 10 are included.

  The eluent liquid feeding mechanism 11 has a plurality of eluent tanks and a pump 1 for feeding the eluent, and further selects an eluent layer in the pump 1 or between the eluent tank and the pump 1. The electromagnetic valve is switched by a signal from the data processing device 10 or a direct input to the pump 1, and the selected eluent is sent by the pump 1. The pump 1 causes the eluent to flow through the flow path 14 at a specified rate in accordance with an analysis program (for example, the program shown in FIGS. 3 and 6).

  The sample introduction unit 3 is a part for injecting a sample (sample) as an analysis target. The sample to be injected contains various components, and the profile of each component is acquired by a chromatogram as described below.

  The eluent is sent to the separation mechanism 12 via the sample introduction unit 3. The separation mechanism 12 includes a guard column 5 and a separation column 6, and the sample introduced from the sample introduction unit 3 passes through the guard column 5 and is separated by the separation column 6. The guard column 5 has a role of removing the impurities contained in the introduced sample and protecting the separation column 6. The separation column 6 separates each component contained in the sample according to their molecular weight. That is, if the molecular weight is small, it is discharged from the separation column 6 earlier in time, and if the molecular weight is large, it takes more time to discharge from the separation column 6 than the component having a low molecular weight.

  The reaction liquid feeding mechanism 13 includes a plurality of reaction liquid layers and a cleaning liquid layer, a pump 2 that feeds the reaction liquid (derivatization reagent) or the cleaning liquid, and a flow path 15. An electromagnetic valve is provided in the pump 2 or between the reaction liquid / cleaning liquid tank and the pump 2. Further, the reaction liquid feeding mechanism 13 switches the electromagnetic valve by a signal from the data processing device 10 or a direct input to the pump 2, and sends the selected liquid to the flow path 15 by the pump 2. The pump 2 also causes the eluent to flow through the flow path 14 at a specified rate in accordance with an analysis program (for example, the program shown in FIG. 2 or FIG. 6) in the same manner as the pump 1.

  The T-shaped joint 7 is provided between the separation column 6 and the reaction liquid feeding mechanism 13, and the reaction liquid and the T-shaped joint in which each amino acid separated by the separation column 6 is fed from the reaction liquid feeding mechanism 13. 7 parts are mixed. This mixed liquid reacts and colors when passing through the reaction device 8 and is detected by the visible detector 9.

  The visible detector 9 outputs a detection signal to the data processing device 10. The data processing device 10 processes the input signal and outputs, records, and stores it as a chromatogram and data.

  In FIG. 1, the data processing apparatus 10 is depicted as being connected only to the visible detector 9, but actually, the eluent liquid feeding mechanism 11, the reaction liquid feeding mechanism 13, and the sample introduction The unit 3, the separation mechanism 12, the reaction device 8, and the visible detector 9 are connected to each other, and a predetermined control process is executed on each of them.

<Content of analysis processing>
FIG. 2 is a flowchart for explaining the overall operation of the analysis processing according to the embodiment of the present invention.
(1) Step S1
(I) The data processing apparatus 10 starts the analysis process according to the input analysis program (see FIG. 3: also referred to as a reference program). The analysis program input in step S1 is a program that prescribes processing by a conventional method, that is, a detection method in which the flow rate of the eluent is constant and the column pressure varies. In the embodiment of the present invention, first, a pressure profile and a chromatogram are obtained in a state where the flow rate of the eluent is constant and the column pressure is varied, and a chromatogram is obtained based on the obtained information, in a state where the flow rate of the eluent is varied and the column pressure is constant. And a new pressure profile is obtained. Once the eluent flow rate setting value is determined and the column pressure can be set to a constant state (a state in which analysis can be performed with the improved analysis program (see FIG. 6)), the column pressure fluctuates over time. The analysis can be continued with the improved program until the pressure resistance of the column is approached. If a change in the pressure over the column over time occurs, the improved analysis program may be adjusted.

(Ii) Analysis Program (FIG. 3: Gradient Program) In the gradient program of the amino acid analyzer of FIG. 3, “% B1” to “% B6” are the ratio of each eluent sent by the pump 1, “flow rate 1”. "Indicates the flow rate that the pump 1 sends. “% R1” to “% R3” indicate the ratio of each reaction solution sent by the pump 2, and “flow rate 2” shows the flow rate sent by the pump 2. A numerical value (that is, 100) corresponding to each time indicates a ratio of supplying the corresponding eluent. That is, only the eluent B1 is supplied from 0.0 minutes to 3.0 minutes, and only the eluent B2 is supplied from 3.1 minutes to 6.9 minutes. The other eluents B3 to B6 are similarly supplied according to the schedule of FIG. The flow rate when each eluent is supplied is constant at 0.400 ml / min as indicated by flow rate 1, and the temperature at that time is maintained at 57 ° C. In the analysis program, the blank part means that the same amount is supplied.

  In FIG. 3, R1 to R3 correspond to prepared reaction liquids or cleaning liquids. And the ratio of reaction liquid R1 and R2 is 50:50, and it supplies from 0.0 minute to 32.0 minutes. From 32.1 minutes to 37.0 minutes, only the reaction solution R3 is supplied, and from 37.1 minutes to 53.0 minutes, the ratio of R1 and R2 is again supplied at 50:50. The flow rate of the reaction solution is constant at 0.350 as shown in the flow rate 2, and the temperature of the reaction solution is maintained at 57 ° C. as in the eluent.

(2) Step S2
The data processing apparatus 10 acquires a chromatogram (S2) according to the input analysis program (FIG. 3). The acquired chromatogram is as shown in FIG. In step S2, the data processing device 10 specifies the order of appearance of each component peak from the acquired chromatogram, and temporarily stores the information in a memory (not shown).

(3) Step S3
The data processing device 10 further acquires a pressure profile (S3). The acquired pressure profile is as shown in FIG.

  As can be seen from FIG. 5, due to the influence of the viscosity of each eluent (B1 to B6) used in the analysis of the amino acid analyzer, the pressure of the pump 1 for sending the eluent varies depending on the type of the eluent to be sent. (The pressure value fluctuates during the gradient program time). However, as described above, the flow rate of each eluent is constant. On the other hand, the pressure of the pump 2 for delivering the reaction liquid is kept constant (the flow rate is also constant).

  In FIG. 2, step S2 is executed first and then step S3 is executed. However, the order in which they are executed may be reversed.

(4) Step S4
The data processing apparatus 10 compares and analyzes the pressure profile (FIG. 5) acquired in step S3 and the pressure resistance reference (maximum value) of the separation column 6 to determine an upper limit pressure set value. That is, the upper limit pressure setting value is determined so that the maximum pressure value of the acquired pressure profile shown in FIG. 5 is, for example, about 80% of the pressure resistance reference of the separation column 6.

(5) Step S5
Based on the fact that the pressure value and the flow rate are in a proportional relationship, the data processing apparatus 10 observes the pressure profile acquired in step S3 and obtains the difference between the pressure value at each time and the upper limit pressure value set in step S4. The flow coefficient value in each time zone of the analysis program input in step S1 is calculated. This calculated flow coefficient value is used to generate an improved analysis program. The flow coefficient value is obtained by, for example, f = P _limit / P _observed . Here, f represents a flow coefficient, P _limit represents a pressure upper limit value, and P _observed represents an actual measurement value given by the pressure profile.

  If the flow coefficient is multiplied for each injection, the retention time varies, so it is desirable to use a constant flow coefficient in a series of analysis sequences. In this case, in order not to always exceed the pressure upper limit value, a flow coefficient that is 80% of the pressure upper limit value (may be 90%. The ratio can be appropriately changed) may be used.

(6) Step S6
(I) Processing contents The data processing apparatus 10 converts the flow coefficient value calculated in step S5 into a flow value. For example, F _NEW = f × F _original can be used as the conversion formula. Here, F _NEW = indicates a new flow value used in the improved analysis program, f indicates the flow coefficient, and F _original indicates the flow value used in the analysis program (FIG. 3).
Further, the flow rate of the reaction liquid on the pump 2 side is also changed using the flow coefficient value calculated in step S5.

(Ii) Improved analysis program By executing step 6, an improved analysis program is generated (see FIG. 6). In the improvement analysis program, the blank part means that the same amount is supplied.

  In FIG. 6, B1 to B6 correspond to the prepared eluent. A numerical value (that is, 100) corresponding to each time indicates a ratio of supplying the corresponding eluent. That is, only the eluent B1 is supplied from 0.0 minutes to 3.0 minutes, and only the eluent B2 is supplied from 3.1 minutes to 6.9 minutes. The other eluents B3 to B6 are similarly supplied according to the schedule of FIG. The flow rate when supplying each eluent is set to 0.450 ml / min from 0.0 minutes to 6.9 minutes, as shown in flow rate 1, and from 7.0 minutes to 14.9 minutes. Set to 0.570 ml / min. Furthermore, after 27.0 minutes, it is set to 0.640 ml / min. The temperature at this time is maintained at 57 ° C.

  In FIG. 6, R1 to R3 correspond to prepared reaction liquids or cleaning liquids. And the ratio of reaction liquid R1 and R2 is 50:50, and it supplies from 0.0 minute to 32.0 minutes. From 32.1 minutes to 37.0 minutes, only the reaction solution R3 is supplied, and from 37.1 minutes to 53.0 minutes, the ratio of R1 and R2 is again supplied at 50:50. The flow rate of the reaction solution is set to 0.400 ml / min from 0.0 minutes to 6.9 minutes, and 0.500 ml from 7.0 minutes to 14.9 minutes, as shown in flow rate 2. Is set to 0.560 ml / min after 15.0 minutes. Further, like the eluent, the temperature of the reaction solution is maintained at 57 ° C.

  In FIG. 6, each flow rate and each mixing ratio are plotted at the leftmost time. Then, these data points may be connected as a line graph to form a set time program (improved analysis program). The flow rate value may be set stepwise, or gradually from the time when a predetermined time has passed (for example, when 3.1 minutes have passed) to the next set value (for example, 0.570 ml / Min) may be linearly changed. The point of change can be determined based on the point of change of the profile in the regions 1 to 3 of the pressure profile (FIG. 3). In addition, the degree of change may be determined based on the amount of change (slope) in the regions 1 to 3. However, even in this case, it is assumed that only the temperature does not form a line graph, but rises suddenly (or falls sharply) stepwise at the changing time. In FIG. 6, a standard analysis method for keeping the temperature constant is assumed, but other methods of amino acid analysis (biological fluid analysis method, hydrolyzate analysis method, etc.) and temperature are detected at predetermined intervals (components to be detected). This is to correspond to the case of changing.

(7) Step S7
The data processing apparatus 10 starts analysis according to the improved analysis program obtained in step 6. In step S7, a chromatogram is acquired.

  FIG. 7 shows a newly acquired chromatogram. As can be seen from FIG. 7, the shape of the obtained graph is the same as the shape of the graph shown in FIG. 4 except for the time scale, but the time at which the peak of each component appears is different from FIG. That is, the analysis time is shortened. Since the appearance order of the peaks of each component is the same as that in the chromatogram of FIG. 4, the component corresponding to each peak can be specified based on the peak information of each component temporarily held.

  In FIG. 7, the appearance time (referred to as holding time) of the peak (vertex) varies by several seconds for each injection. In order to absorb this variation, a time width (time window) of about several tens of seconds is generally set. In the embodiment of the present invention, the flow rate setting is switched so that the pressure is in the vicinity of the increase. If a peak appears at this flow rate switching point, the holding time may change significantly. In order to prevent this, it is important to switch the flow rate while avoiding the time zone when the peak appears.

(8) Step S8
The data processing apparatus 10 acquires a pressure profile (FIG. 8).
As shown in FIG. 8, the pressure profile obtained by the improved analysis program is substantially constant for both pumps 1 and 2. The pressure of the pump 2 is detected by a sensor located immediately after the pump (liquid discharge side). The reason why the pressure does not fluctuate even though the flow rate of the pump 2 is changed is that the pump 2 supplies the reaction liquid whose ratio to the amount of the fluid on the flow path is very small. This is because there is no significant change in the pressure in the flow path even if it is changed.

(9) Step S9
As described above, ideally, the gradient elution program is adjusted so that the analysis pressure is in the vicinity of the upper pressure limit of 80%. Moreover, the mixing ratio of the mobile phase and the reaction liquid can be made constant by setting the flow rate of the pump 2 to be substantially proportional to the flow rate of the pump 1. However, the pressure profile during analysis may vary due to changes in factors such as flow rate, mobile phase viscosity, column temperature, and the like. Steps S9 and S10 are performed to adapt to this variation.

  The data processing device 10 determines whether the pressure value given by the pressure profile acquired in step S8 is 90% or more of the pressure upper limit value determined in step S4. If the pressure value given by the pressure profile is within 90% of the pressure upper limit value, the process proceeds to step S7, and the analysis is continued using the same conditions (same improved analysis program). On the other hand, when the pressure value given by the pressure profile is 90% or more of the pressure upper limit value, the process proceeds to step S10.

(10) Step S10
The data processing device 10 calculates the flow coefficient value again using the current pressure value given from the pressure profile obtained in step S8.

  And a process transfers to step S6 again, a flow volume setting value is updated, and a new improvement analysis program is produced | generated. Since the processing contents are the same as those in step S6, they are omitted.

(11) Others When analyzing using the same conditions (using the same eluent and reaction solution) and the same improved program, the analysis program input in step S1 is changed to the improved analysis program, and the processing of steps S2 to S6 is performed. Can be omitted. In this case, it is necessary to include the process of step S6 in the process of step S10.

<Others>
(I) In this embodiment, an improved analysis program is given to the analysis apparatus. The improved analysis program is created based on the reference program, but may be created in a series of analysis processes, or may be created in advance and given during actual analysis. This improved analysis program changes the flow rate of the eluent over time (stepwise or temporarily changes) and introduces it into the first flow path to realize the process of keeping the pressure of the liquid applied to the separation mechanism within a predetermined range. It is a program to do. Based on this improved analysis program, a data processing device (also referred to as a computer, a processor, etc.) in the analyzer controls the flow rate of the eluent by operating the liquid feeding operation of the first pump, and the detection signal from the reaction device Are used to obtain a pressure profile indicating the change over time of the pressure applied to the separation mechanism and a chromatogram of the mixed solution. By doing so, the pressure profile is controlled to be substantially constant, and the analysis result can be acquired in a shorter time than in the past (in the case of constant flow rate and pressure fluctuation).

  Further, the improved analysis program stipulates that the flow rate of the reaction solution (derivatizing reagent) varies in accordance with the change in the flow rate of the eluent. Therefore, according to the improved analysis program, the liquid feeding operation of the second pump is operated and the flow rate of the reaction liquid (derivatized reagent) is also controlled. In this way, the mixing ratio of the reaction solution and the eluent can be kept constant, and the baseline can be stabilized. Here, the baseline means the baseline of the chromatogram (shows that the Y axis other than the peak in FIGS. 4 and 7 is zero). Also, “stabilization” means that the noise on the baseline is suppressed to show a flat state.

  The improved analysis program is generated as follows. That is, based on a reference program in which the flow rates of the eluent and the induction or reagent are kept constant and sent to the first and second flow paths, respectively, and as a result, the pressure of the liquid applied to the separation mechanism varies. To obtain the required reference pressure profile and reference chromatogram. Then, the flow rate is calculated so that the temporal pressure value indicated by the reference pressure profile falls within a predetermined range (so that the pressure profile is substantially constant as shown in FIG. 8), and the improved analysis program is obtained. More specifically, first, an upper limit pressure value to be applied to the separation mechanism is determined, and a ratio between the upper limit pressure value and each pressure value of the eluent indicated by the reference pressure profile is obtained as a flow coefficient. Further, the fluctuation flow rate of the eluent defined by the improved analysis program is determined by multiplying the constant flow rate indicated in the reference program by a flow rate coefficient. By doing so, it is possible to generate an improved analysis program that guarantees a reduction in damage to the separation mechanism (column).

  Furthermore, in this embodiment, even if the analysis is once started using the improved analysis program, if the pressure profile fluctuates during the process (when the almost constant state cannot be maintained), the eluent in the improved analysis program is changed. Change the flow rate and update the improved analysis program. More specifically, a ratio between an upper limit pressure value to be applied to the separation mechanism and each pressure value of the eluent indicated by a pressure profile obtained from the improved analysis program is obtained as a flow coefficient. Then, the fluctuation flow rate of the eluent in the improved analysis program is updated by multiplying the given reference flow rate by the flow coefficient. By doing so, minute fluctuations caused by repeated analysis can be corrected, so that it is possible to ensure that a more accurate analysis result is obtained.

(Ii) The present invention can also be realized by software program codes that implement the functions of the embodiments. In this case, a storage medium in which the program code is recorded is provided to the system or apparatus, and the computer (or CPU or MPU) of the system or apparatus reads the program code stored in the storage medium. In this case, the program code itself read from the storage medium realizes the functions of the above-described embodiment, and the program code itself and the storage medium storing the program code constitute the present invention. As a storage medium for supplying such program code, for example, a flexible disk, CD-ROM, DVD-ROM, hard disk, optical disk, magneto-optical disk, CD-R, magnetic tape, nonvolatile memory card, ROM Etc. are used.

  Also, based on the instruction of the program code, an OS (operating system) running on the computer performs part or all of the actual processing, and the functions of the above-described embodiments are realized by the processing. May be. Further, after the program code read from the storage medium is written in the memory on the computer, the computer CPU or the like performs part or all of the actual processing based on the instruction of the program code. Thus, the functions of the above-described embodiments may be realized.

  Further, by distributing the program code of the software that realizes the functions of the embodiment via a network, it is stored in a storage means such as a hard disk or memory of a system or apparatus, or a storage medium such as a CD-RW or CD-R And the computer (or CPU or MPU) of the system or apparatus may read and execute the program code stored in the storage means or the storage medium when used.

  Finally, it should be understood that the processes and techniques described herein are not inherently related to any particular apparatus, and can be implemented by any suitable combination of components. In addition, various types of devices for general purpose can be used in accordance with the teachings described herein. It may prove useful to build a dedicated device to perform the method steps described herein. Various inventions can be formed by appropriately combining a plurality of constituent elements disclosed in the embodiments. For example, some components may be deleted from all the components shown in the embodiment. Furthermore, constituent elements over different embodiments may be appropriately combined. Although the present invention has been described with reference to specific examples, these are in all respects illustrative rather than restrictive. Those skilled in the art will appreciate that there are numerous combinations of hardware, software, and firmware that are suitable for implementing the present invention. For example, the described software can be implemented in a wide range of programs or script languages such as assembler, C / C ++, perl, shell, PHP, Java (registered trademark).

Furthermore, in the above-described embodiment, control lines and information lines are those that are considered necessary for explanation, and not all control lines and information lines on the product are necessarily shown. All the components may be connected to each other.
It should be noted that the specification and specific examples are merely typical, and the scope and spirit of the present invention are set forth in the following claims.

  DESCRIPTION OF SYMBOLS 1,2 ... Pump, 3 ... Sample introduction part, 5 ... Guard column, 6 ... Separation column, 7 ... T-joint, 8 ... Reactor, 9 ... Visible detector, 10 ... Data processor, 100 ... Amino acid analyzer

Claims (14)

An analyzer for acquiring a chromatogram of a liquid sample,
A first pump for feeding the eluent;
A second pump for delivering a derivatizing reagent;
A first flow path extending from the first pump;
A second flow path extending from the second pump;
A sample introduction part for introducing a sample to be analyzed, which is provided in a stage subsequent to the first pump in the first flow path;
A separation mechanism for separating each component of the sample provided in a stage subsequent to the sample introduction unit in the first flow path;
A joint part provided at a subsequent stage of the separation mechanism, connecting the second channel to the first channel, and mixing the sample and the derivatization reagent;
A detector for detecting a reaction between the sample and the derivatizing reagent and outputting a detection signal;
A data processing device that controls each component of the analyzer and generates and outputs a chromatogram based on the detection signal;
The data processing apparatus includes a given analysis program for changing the flow rate of the eluent over time and introducing it into the first flow path to keep the pressure of the liquid applied to the separation mechanism within a predetermined range. Based on this, the flow profile of the eluent is controlled by operating the liquid feeding operation of the first pump, the pressure profile indicating the change over time in the pressure applied to the separation mechanism from the detection signal, and the chromatogram of the liquid mixture The analysis apparatus characterized by acquiring. In claim 1,
The given analysis program stipulates that the flow rate of the derivatizing reagent varies in response to variations in the flow rate of the eluent,
The data processing apparatus controls the flow rate of the derivatized reagent by operating a liquid feeding operation of the second pump based on the given analysis program. In claim 1,
The data processing device keeps the flow rates of the eluent and the induction or reagent constant and sends them to the first and second flow paths, respectively, and as a result, the pressure of the liquid applied to the separation mechanism varies. The necessary reference pressure profile and reference chromatogram are obtained based on the reference program to be calculated, and the flow rate is calculated so that the pressure value with time indicated by the reference pressure profile falls within the predetermined range. An analyzer characterized by generating a program. In claim 3,
The data processing device determines an upper limit pressure value to be applied to the separation mechanism, obtains a ratio between the upper limit pressure value and each pressure value of the eluent indicated by the reference pressure profile as a flow coefficient, and the reference program A variable flow rate of the eluent defined by the analysis program is determined by multiplying the constant flow rate shown in FIG. In claim 1,
The data processing device is characterized by changing the flow rate of the eluent in the analysis program and updating the analysis program when the magnitude of the fluctuation of the pressure profile obtained from the analysis program is a predetermined value or more. Analysis equipment. In claim 5,
The data processing device obtains, as a flow coefficient, a ratio between an upper limit pressure value to be applied to the separation mechanism and each pressure value of the eluent indicated by a pressure profile obtained from the analysis program. An analytical apparatus characterized by updating a fluctuation flow rate of the eluent in the analysis program by multiplying by a flow rate coefficient. In claim 1,
The analysis program is a program for changing the flow rate of the eluent stepwise or changing the flow rate of the eluent in an inclined manner. A method for obtaining a chromatogram of a liquid sample using an analyzer,
The analyzer includes a first pump for feeding an eluent, a second pump for feeding a derivatization reagent, a first flow path extending from the first pump, and the second A second flow path extending from the first pump, a sample introduction section for introducing a sample to be analyzed, which is provided in a stage subsequent to the first pump in the first flow path, and the first flow path. A separation mechanism that separates each component of the sample provided at the subsequent stage of the sample introduction unit, and a second stage that is provided at the subsequent stage of the separation mechanism and connects the second flow path to the first flow path And a joint unit for mixing the sample and the derivatizing reagent, a detector for detecting a reaction between the sample and the derivatizing reagent and outputting a detection signal, and each component of the analyzer. A data processing device for generating and outputting a chromatogram based on the detection signal , Has a,
The method
The data processing device controlling the flow rate of the eluent by operating the liquid feeding operation of the first pump based on a given analysis program;
The data processing device comprises, from the detection signal, a pressure profile indicating a change with time of the pressure applied to the separation mechanism, and a step of acquiring a chromatogram of the mixed solution,
The analysis program is a program for changing the flow rate of the eluent over time and introducing it into the first flow path to keep the pressure of the liquid applied to the separation mechanism within a predetermined range. Method. In claim 8,
The given analysis program stipulates that the flow rate of the derivatizing reagent varies in response to variations in the flow rate of the eluent,
The data processing apparatus further comprises a step of controlling a flow rate of the derivatizing reagent by operating a liquid feeding operation of the second pump based on the given analysis program. In claim 8,
Further, the data processing device keeps the flow rates of the eluent and the induction or the reagent constant and sends them to the first and second flow paths, respectively. As a result, the pressure of the liquid applied to the separation mechanism is increased. Based on the reference program that will fluctuate, obtain the required reference pressure profile and reference chromatogram, calculate the flow rate so that the pressure value over time indicated by the reference pressure profile falls within the predetermined range, A method comprising generating the analysis program. In claim 10,
In the step of generating the analysis program, the data processing device determines an upper limit pressure value to be applied to the separation mechanism, and a ratio between the upper limit pressure value and each pressure value of the eluent indicated by the reference pressure profile. Is determined as a flow coefficient, and the constant flow indicated in the reference program is multiplied by the flow coefficient to determine the fluctuating flow rate of the eluent defined by the analysis program. In claim 8,
Furthermore, when the magnitude of the pressure profile fluctuation obtained from the analysis program is greater than or equal to a predetermined value, the data processing device changes the flow rate of the eluent in the analysis program and updates the analysis program. A method characterized by comprising. In claim 12,
In the updating step, the data processing device obtains a ratio between an upper limit pressure value to be applied to the separation mechanism and each pressure value of the eluent indicated by a pressure profile obtained from the analysis program as a flow coefficient. A variable flow rate of the eluent in the analysis program is updated by multiplying the determined reference flow rate by the flow coefficient. In claim 8,
The analysis program is a program for changing the flow rate of the eluent stepwise or changing the flow rate of the eluent in an inclined manner.

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