JPH049756A - Liquid chromatography analysis system - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a liquid chromatography analysis system, and particularly to a liquid chromatography analysis system suitable for continuous automatic analysis of a plurality of samples.

[Prior Art] Liquid chromatography is characterized by its ability to separate components in an eluted state and then selectively analyze specific components separated by chemical reactions, etc., and is an important analytical means in the field of clinical testing. However, the analytical methods are complex and require skill to operate, and the analysis takes time, which reduces the number of samples to be analyzed in routine work such as clinical tests where a large number of samples must be analyzed within a limited time. This is hindering the increase in the number of liquid chromatographs, and is the reason why the spread of liquid chromatography analyzers is delayed.

Regarding conventional analysis methods using liquid chromatographs for medical use, we will explain the analysis method for blood catecholamines as a specific example6. It has been recognized to be effective in diagnosis, and is considered an important item in clinical tests such as comprehensive examinations for adult diseases. The analytical method is to separate the three components epinephrine, norepinephrine, and dopamine using a liquid chromatograph, react them with a fluorescent labeling agent, and measure the concentration of each component using a fluorometer.6 No. 13
The figure shows a flow path diagram of a typical conventional catecholamine analyzer.

In the autosampler 1, plasma samples that have been previously subjected to protein removal treatment using a centrifuge or the like are stored and arranged in a sample container 2. The sample is drawn through a nozzle 4 by a pump 3 and introduced into a sample loop 6 by a sample injector 5.

On the other hand, a pretreatment liquid 9 is supplied to the precolumn 7 by a pump 8 through a valve 10. The eluent 14 flows through the sample injector 5 and the column switching valve 11, and the eluent 14 flows into the separation column 12 by the pump 13 via the valve 15 and the column switching valve 11.

By switching the sample injector 5 in this state, the sample in the sample loop 6 is sent to the precolumn 7 by the pretreatment liquid 9, where it is adsorbed and concentrated by the adsorbent in the column, and impurities are removed by the pretreatment liquid 9. It is discharged from the discharge pipe 16.

Next, when the column switching valve 1 is switched, the eluent 14 flows through the pre-column 7, the sample adsorbed on the pre-column is eluted and sent to the separation column 12 for separation, and the eluted sample is transferred to the pump 17. While passing through a reaction coil 19, labeling with a fluorescent agent is performed, and the concentration of each component is measured using a fluorometer 20.

The cleaning liquid 21 can be sent to the reaction coil 19 to clean it by switching the valves 22, 23 at the appropriate times.

In catecholamine analysis using the conventional method described above, it takes about 40 minutes to analyze one sample, which inevitably limits the number of samples to be analyzed.

In addition, before starting an analysis, it is necessary to bleed air from the tube between the eluent tank and the pump, warm up to stabilize each device, and also to judge whether the chromatograph is normal or not. Equipment maintenance and column life determination require considerable experience and high skill. Therefore, even if they are equipped with expensive measuring equipment, it is not easy to train operators, and many of the laboratories in general hospitals and the like currently request analysis to specialized analysis and testing centers.

[Problems to be Solved by the Invention] In the conventional analysis of catecholamines, (1) a reaction solution is combined with a sample separated and eluted in a separation column, and fluorescent labeling is performed in a reaction coil. Therefore, the sample and reaction solution mix as they flow through the reaction coil, and the reaction progresses, but the reaction takes a certain amount of time (3
Not only does it take about 5 minutes), but also the sample is diluted by the reaction solution, which significantly reduces the degree of separation of the sample.

Therefore, it is necessary to use a separation column with a large capacity that allows for a decrease in resolution, which naturally causes the problem of longer analysis time.

(2) Also, determine whether or not the pump, detector, thermostatic chamber, and other parts of the equipment have been sufficiently warmed up before starting measurement.
Judging the presence or absence of measurement samples, labeling reagents, eluents, etc.
There are problems in that a skilled operator is required to judge column life (separation ability), manage daily fluctuations in standard samples, etc., and be able to respond immediately when the system goes down.

An object of the present invention is to improve the automation and operability of a liquid chromatography analysis system. Another object of the present invention is to provide an automatic analysis system that can continuously and efficiently analyze a large number of samples.

[Means to solve the problem] The gist of the present invention to achieve the above object is as follows.

A liquid chromatography analysis system for analyzing liquid components, the system includes an autosampler section, an analysis section, a control section, and various sensors, the analysis section includes a precolumn, a separation column, a detector, and a data processing device, and the system includes: Various sensors are arranged in the operating parts of the autosampler section, the analysis section, and the control section, and the autosampler section has a sample preparative distribution means, a reagent addition means, and a sample derivatization reaction processing means, The pre-column has the function of adsorbing and concentrating the sample introduced from the autosampler section, the separation column has the function of separating the derivatized sample concentrated by the pre-column and eluted into the eluent, and the detector has the function of separating and concentrating the sample introduced from the autosampler section. The data processing unit includes means for measuring the concentration of each component of the derivatized sample separated by the column, and the data processing unit performs arithmetic processing on the measurement data of the concentration;
The control unit has a function of calculating and processing the detection signals of the respective sensors and outputting and storing related data, and the control unit controls and analyzes each of the means based on a preset program and the calculation results of the data processing unit. A liquid chromatography analysis system is characterized by the following features:

As the autosampler, use one that has a preprocessing function that allows processing such as sample collection, distribution, reagent addition, mixing, and reaction on the sample table, and before introducing the sample into the analysis section, It is possible to perform derivatization such as fluorescent labeling.

Therefore, the sample eluted from the separation column does not need to pass through a reaction coil as in the conventional example, so it can be measured at the shortest position from the outlet of the separation column without impairing the separation. Therefore, the separation column does not need to take into account the degradation of separation in the reaction coil after elution as in conventional examples, so the capacity of the column can be reduced and the separation time can be shortened. In addition, since there is no need to pass through a reaction coil, the processing time after the first separation column can be significantly shortened.

Furthermore, in order to shorten the analysis time, it is necessary to shorten the analysis time in the separation column, which occupies most of the analysis time.

This can be done by shortening the column length of the separation column or by reducing the column diameter.

Although the separation capacity of the column decreases when the column length is shortened or the column diameter is made thin, the separation capacity can be increased by reducing the particle size of the packing material.

In addition, in order to realize high-speed analysis, for example, it is necessary to
mm x 5mm length separation column and 2 particle size 1.5μm
This can be achieved by using a packing material of

The analytical processing capacity (sampling interval) can be estimated using the following formula.

σc””VR”/N σc2: band spread in column V-K: Retention volume N: Number of theoretical plates of column VR=(k’+1)V.

k゛: Capacity ratio ■o: Column void volume V, = επr2L ε: Column porosity r: Column radius L: Column length N=L/H H Theoretical plate height of two columns σ4 (t )≦0.32σC(t) σ<(t): Detector sampling interval σc(1): Time spread in column σo(1)=σ. /FF: For flow rate theoretical packing, H=2d.

d: Particle size of the packing material and, in general, the sampling interval of the detector is calculated using the peaks that are not retained on the column.

k'=o, F=1.0mA/win, 1=Q, 5°d,
In the case of a column with =1.5 μm, r=0.5 mm, and L=5 mm, σ4(t) is 61.3 m5ec.

The above is from the Journal of Chromatography.
Library Volume 28 Microcolumn Biperformance Liquid Chromatography (1984)
Pages 1 to 38 (Journal of Chromatography
y Ljbrary volume 28: Micro
column lligh-Performance
Liquid Chromat.

graphy (1984) PP, 1-38),
and Journal of Chromatography Library, Volume 32: The Science of Chromatography (1985), pp. 435-447.
y Library volume 32 :Th
e 5science of Chromatograph
hy (1985) PP, 435-447).

Therefore, in the present invention, high-speed analysis can be achieved by using the pre-label method, the micro-separation column method, and high-speed data processing with a sampling interval of 1 m5 ec.

In addition, in order to realize one-touch start automatic analysis, we also check the presence or absence of the sample, the amount of reagent and eluent, the temperature,
Equipped with self-diagnosis functions such as pump pressure, water leak detection, detector lamp output, noise or drift amount display, etc.It also displays the sample number currently being analyzed, the number of remaining samples, and the analysis end time. It was designed to allow

It also performs data processing such as determining the lifespan of columns, measuring daily fluctuations in reagents and standard samples, managing various measurement data, and comparing with past data stored in memory.

In order to achieve the above-mentioned high-speed analysis and automatic analysis, a semiconductor memory with a fast operation of 1 megabit or more or a CPU with 32 bits or more and an internal memory of 2 megabytes or more is required.
it: central processing unit @) is suitable for performing high-speed data processing.

The autosampler used is one that can perform pretreatment operations such as sample fractionation, distribution, reagent addition, and one reaction on the sample table.

The precolumn in the analysis section introduces and adsorbs the sample introduced from the sample injector, thereby concentrating the sample and removing impurities. The concentrated sample is eluted with an eluent and sent to a separation column using a column flow switching valve.

The separation column separates the specific components of the sample concentrated by the pre-column.

The detector sequentially introduces the sample separated by the separation column into, for example, a fluorometer having a flow cell, and measures the concentration of the sample.

The various sensors check the operation of each device, detect the presence or absence of a sample on the autosampler, the pressure of each pump, the temperature of the sample cooling device, the temperature of the reaction vessel constant temperature bath, the temperature of the column constant temperature bath, and detect water leakage inside the device. , measure the sample volume, eluent volume, reagent volume, etc.

The data processing section calculates measurement results, diagnoses each device from the detection signals and measurement results of various sensors, handles abnormalities, determines column life, and processes eluent.

Performs various data processing such as managing the quality and volume of reagents and standard samples, calculating and storing measured data, and comparing with past data.

The main purpose of the present invention is automatic analysis with a one-touch start, and the analysis is performed in the following steps.

Sample containers containing samples to be analyzed are arranged in advance in the autosampler, and a sample derivatization reagent is prepared on the autosampler as necessary.

When the start button is pressed, the operation of the autosampler, pump, reaction tank, column constant temperature bath, detector, data processing device, sample volume, eluent volume, etc. are checked (system self-diagnosis).

Check the conditions of the above-mentioned devices, sample, and eluent, and if these are normal, start warming up. If there is an abnormality, a message indicating the abnormality location and its situation will be displayed on the CRT (cathode ray tube) screen and the abnormality will be corrected.

The determination to start analysis is made after, for example, measuring the noise value or drift value of the detector during warming up and confirming that the measured value is below a predetermined value (hereinafter, this is referred to as a test run). .

If the values of noise and drift do not fall below predetermined values, the values are displayed on the CRT and the program is programmed so that confirmation measurement is performed again after a certain period of time. It is programmed to automatically start analysis after confirming the normal operation of each device and completing the test run.

When analysis starts, the autosampler's dispensing nozzle operates.
Inhale a predetermined amount of the sample in the sample container.

At this time, the presence or absence of air in the dispensing nozzle or the presence or absence of a sample container is detected (hereinafter, this operation is referred to as a blank sample test). As the sensor, for example, an optical sensor using an LED (light emitting diode) or the like is suitable.

For example, if it is determined that there is no sample in the blank sample test, abnormal processing is performed to stop the analysis and display this on the CRT. In addition, if it is determined that there is a sample, the sample is dispensed into another empty container prepared on the autosampler, and then a predetermined amount of derivatization reagent is inhaled and dispensed 9 times to mix the sample and derivatization reagent. let When dispensing the derivatization reagent, a blank sample test is performed in the same way as when dispensing samples. The above derivatization treatment may be performed using another container, such as a reaction coil. It is desirable that the derivatization treatment be performed while being maintained at a predetermined temperature.

Next, the derivatized sample is introduced into the analysis section. When derivatized in a reaction container on an autosampler, the sample is sucked into a dispensing nozzle, injected into a sample loop of a sample injector, and introduced into a precolumn by switching the flow path. Derivatization can also be carried out within the sample loop of the sample injector.

A pretreatment liquid prepared in advance to be suitable for sample concentration and impurity removal is fed to the precolumn by a pump and flows at a constant flow rate. The sample introduced by the sample injector is adsorbed and concentrated by the precolumn, and impurities are discharged from the outlet below the precolumn by the pretreatment liquid.

An eluent prepared to properly separate the sample is also supplied to the separation column by a pump and flows at a constant flow rate. By switching the column switching valve for introducing the sample, the eluent elutes the sample concentrated in the pre-column and introduces it into the separation column, whereby each component in the sample is separated. When the sample has completely transferred from the pre-column to the separation column, the column flow switching valve is switched to allow the eluent to flow directly into the separation column to continue sample separation.

On the other hand, the pre-treatment liquid is poured into the pre-column again to regenerate it so that the next sample can be introduced.

The sample separated by the separation column is introduced into a flow cell of a detector, and the concentration of sample components is measured. The measurement data is processed by the CPU and displayed on a CRT or output to a printer. Furthermore, the processed data is written to the memory section.

The data written in the memory section is fed back during self-diagnosis of each unit and the next data processing, and is also used for backup when starting up the next analysis. Furthermore, it is also possible to perform a comparative evaluation with past data written in the memory section.

The series of operations described above are preprogrammed and are automatically performed by simply pressing the start button. Therefore, a particularly skilled operator is not required.

Further, it is also possible to automatically perform the desired analysis by simply inserting an IC card into which necessary analysis items, etc., are entered in the analysis menu.

[Function] In the analysis system of the present invention, self-diagnosis and abnormality processing of each component device, management of analytical reagents, etc. can be performed by configuring a series of analysis systems using a CPU,
The reason is that the analysis is performed using preprogrammed procedures and arithmetic processing by the CPU.

This achieves one-touch operation,
Routine work such as clinical tests that aim to rapidly process a large number of samples can also be performed without the need for a particularly skilled operator.

[Example] As an example of the liquid chromatography analysis system of the present invention, an analysis example of catecholamines will be explained.

Figure 1 shows a system diagram of the catecholamine analysis system.

This system consists of an autosampler 1 and a data processing device 6.
3 and an analysis section 49.

A sample rack 31 is attached to the sample stage 32 of the autosampler 1, and the sample rack stores various sample containers 2, a reaction reagent 18 for fluorescent labeling, an internal standard solution 35, and a standard sample 36 in their respective containers. It has been fitted. Samples such as plasma and urine are used that have been previously subjected to protein removal treatment.

In addition, a reaction container 37 is located near the sample stage 32,
Nozzle cleaning tank 38. Drain boat 39° Injection boat 4
0 is attached. The drive mechanism 41 of the nozzle 4 is
, z-direction, and can move the nozzle 4 vertically, horizontally, vertically, and vertically to move the sample onto each container or each boat.

The nozzle 4 is connected to a dispensing pump 45 and a container for the cleaning liquid 21 via a three-way valve 44 with a pipe 43 made of fluororesin or the like. The tube 43 is equipped with a detector 47 for detecting an empty sample using a light emitting diode or the like.

The dispensing pump 45 uses a syringe pump driven by a pulse motor. The reaction container constant temperature bath 48 is the reaction container 37
The sample stage 32 is equipped with a temperature sensor to maintain the temperature at a predetermined temperature, and the sample stage 32 is equipped with a cooling device, so that the temperature is controlled by the temperature sensor and the control device so that the samples and reagents on the sample rack 31 can be kept at a low temperature. The analysis section 49 consists of a pre-column system for concentrating the sample and removing impurities, a separation column system for separating sample components, a detector, and a data processing section.

In the pre-column system, the pre-treatment liquid 9 is fed at a constant flow rate by a pump 51 and flows into the pre-column 7 via the sample injector 5. The sample injector 5 is provided with a sample loop 6 that measures a predetermined amount of sample injected from the injection port 4o of the autosampler 1 and introduces the sample into the analysis section. The pre-column constant temperature bath 55 maintains the pre-column 7 at a predetermined temperature.

In the separation column system, the eluent 14 is fed at a constant flow rate by the pump 57 and flows into the separation column 12 via the column switching valve 11. By switching the column switching valve 11, the eluent flows through the precolumn 7 and transfers the sample processed in the precolumn to the separation column 12. The separation column constant temperature bath 60 maintains the separation column 12 at a predetermined temperature.

The detector 61 is a fluorometer that measures the fluorescence intensity of sample components eluted from the separation column 12 and has a flow cell 62 .

The data processing unit includes a data processing unit @ 63 that performs calculation processing on the measurement results of the detector 61, a calculation processing of measurement values of various sensors,
It includes a CPU 64 for controlling memory and each device, an output printer 5, a CRT 66, and the like. Note that the switching valve 68 is provided so that the liquid in the pumps 51, 57 can be discharged as necessary, and is set to be automatically discharged before the start of warming-up.

The analysis according to this embodiment is performed in the following order: (1) Diagnosis of the status of each device (initialization) (2) Warming-up (2) Measurement of standard sample for analysis (3) Measurement of unknown sample (3) Data processing. Below, they will be explained in order.

(2) Status diagnosis (initialization) of each device FIG. 2 shows a flowchart for diagnosing the status of each device.

Each device starts operating by pressing the start button,
Checks the ROM, etc. in the CPU 63, and if there is an abnormality, displays a message such as "rC: PU abnormal" on the CRT 66,
``Abnormality processing'' is performed to stop each device (hereinafter, abnormality processing means displaying an abnormality and stopping each device).

If normal, the data processing device 63 is checked. If the data processing device is abnormal, abnormality processing is performed.

If the data processing section is normal, the autosampler, pump, and valve switching device are checked, and if they are abnormal, abnormality processing is performed. In the normal case, a warm-up process is performed.

In this example, the autosampler, pump, and valve switching device each have a built-in CPU, but
It may also be controlled by the CPU 64. Further, in the case of abnormality processing, in addition to the abnormality display, a countermeasure against the abnormality may be displayed, and the abnormality processing may be performed by the operator.

■ Warming-up process FIG. 3 shows a flowchart of the warming-up process.

First, the number of sample containers accommodated on the sample rack 31 of the autosampler 1 is measured.

The number of sample containers is measured by driving a contact sensor 69 attached to the nozzle 4 shown in FIG. 4 by a drive mechanism 41. Note that a contact sensor 70 may be provided at the bottom of the sample container 33 for measurement. If you forget to set the sample container, a message such as ``No sample, please set r sample'' will be displayed on the CRT 66, and abnormality processing will be performed. The number of sample containers measured is stored in the CPU and displayed on the CRT 66.

Next, the amount of eluent is measured using a weight sensor, a liquid level sensor, or the like. The data processing unit 63 calculates the number of samples that can be analyzed based on the measured amount of eluent, compares it with the number of samples calculated from the number of sample containers, and if the amount of eluent is insufficient, it indicates "insufficient eluent." After replenishing the eluent, a message such as "Please press the start button" will be displayed on the CRT66. After replenishing the eluent, press the start button to measure the eluent volume again and calculate the number of samples that can be analyzed.

The same operation is performed for the pretreatment liquid. In addition, the same operation is performed for the surplus amount of waste liquid.

If the eluent volume and pretreatment fluid volume are sufficient for analysis, then the pressure of the pump 51.57 is detected by a pressure sensor, and the value is within a preset pressure range. Determine whether or not. If the measured pressure is outside the set pressure range, all devices are stopped, a message such as "Pump pressure abnormality" is displayed on the CRT 66, and abnormality processing is performed. In this case, the abnormal location can be determined by switching the valve 11.

If the pressure is normal, the temperatures of the sample cooling device 3 in the sample rack, the pre-column constant temperature bath 55, the separation column constant temperature bath 60, and the reaction vessel constant temperature bath 48 are measured and are determined to be within a preset temperature range. Determine whether or not. If the temperature is outside the set range, a message such as "precolumn constant temperature chamber abnormal" is displayed on the CRT 66, and abnormality processing is performed.

In addition, if the measured temperature is higher than the set range, in addition to displaying "Thermostatic chamber abnormality", all equipment other than the pump will be stopped to protect the column, and the pump will be turned off after the temperature has decreased. Program it to stop. When the temperatures of the pre-column constant temperature bath 551, the separation column constant temperature bath 60, and the reaction vessel constant temperature bath 48 are normal, a test run is performed to measure noise and drift for a certain period of time, and it is determined whether or not analysis is possible. Furthermore, by monitoring the baseline level of the detector, the intensity of the detector lamp can be determined, and the lamp life can be determined.

If the noise value and drift value in the test run are larger than the set value, the test run is performed again after a certain period of time, and when the noise value and drift value are within the set value, analysis is automatically started.

■ Analysis FIG. 5 shows a flowchart of the analysis.

(1) Move the reaction vessel cleaning nozzle 4 to the position of the reaction vessel 37, and
5 to inject the cleaning liquid 21. The injection amount is larger than the capacity of the reaction container, and the excess cleaning liquid is overflowed and discharged from the drain boat 39. The above operation is repeated several times (for example, three times) to clean the reaction container 37.

(2) Move the sample suction nozzle 4 to the position of the sample container 2 to be analyzed, and suction a predetermined amount of sample. Further, at this time, in order to correct fluctuations in the recovery rate of the entire device such as the column, a step may be added in which an internal standard solution (isoproterenol in this example) is sucked in in the same manner.

(3) After inhaling the empty sample test sample, the pump 45 is stopped, and the presence or absence of air in the tube 43 is checked by the empty sample test detector 47 that monitors changes in light intensity using an LED.

If there is air, the nozzle 4 is moved to the drain boat 39 and the cleaning liquid is discharged several times to expel the air inside the nozzle. Next, abnormality processing is performed to display a message such as "No sample" on the CRT 66. If there is no air, "Sample Confirmation" is displayed on the CRT 66, and then the process moves to the next operation.

(4) Move the sample discharge nozzle 4 to the position of the reaction container 37 and dispense the sucked sample into the reaction container 37.

(5) Nozzle cleaning After moving the nozzle 4 to the drain boat 39 and discharging the cleaning liquid to wash out the sample adhering to the nozzle inner wall, move it to the nozzle cleaning tank 38 and lower it into the tank to discharge the cleaning liquid. Clean the outside of the tip of the discharge nozzle.

(6) Move the reagent inhalation nozzle 4 to the position of the reaction reagent 18 and inhale a predetermined amount of the reagent.

(7) Empty sample test After inhaling the reaction reagent, the pump 45 is stopped and the presence or absence of air in the tube 43 is checked by the empty sample test detector 47. If there is air, the nozzle 4 is moved to the drain boat 39, and the cleaning liquid is Expel the air in the nozzle by performing the discharge stroke several times, and then replenish the reaction reagent and press the start button.
etc. and performs abnormality processing. After replenishing the reaction reagent,
When the start button is pressed, reagent inhalation and blank sample testing are performed again. If there is no air, display "Reaction reagent confirmation" on the CRT 66 and move on to the next operation.

(8) Move the reagent discharge/mixing nozzle 4 to the position of the reaction container 37, inject the inhaled reaction reagent into the reaction container, and mix it with the previously injected sample. Mixing methods include air discharge, external vibration, stirring, etc., but if it is a low viscosity liquid, it can be mixed by high speed discharge.

(9) Reaction The mixture of the above sample and reaction reagent is reacted in a reaction container for a predetermined period of time to derivatize the sample.

(10) Sample introduction The sample derivatized product after the reaction is inhaled through the nozzle 4, inserted into the injection boat 40, and injected into the sample loop 6 of the sample injector 5.

Next, when the sample injector 5 is switched, the sample derivatized product is sent to the precolumn 7 by the flow of the pretreatment liquid.

(11) The sample derivatized product is adsorbed and concentrated in the concentration precolumn, and impurities that interfere with analysis are discharged from discharge lower 0.

(12) When the sample transfer column switching valve 11 is switched, the eluent flows through the pre-column 7, and the sample derivatized product concentrated in the pre-column is dissociated and sent to the separation column 12 to start separation. When all the sample derivatized products have been transferred to the separation column 12, the column switching valve 11 is switched again (the state shown in FIG. 1), and the eluent flows directly to the separation column 12 without passing through the precolumn 7. Pretreatment liquid flows.

(13) Separation Subsequently, the eluent is passed through the separation column 12 to separate the target sample derivatized product.

(14) Pre-column regeneration In parallel with the separation of the sample derivatized product, the pre-treatment liquid is passed through the pre-column 7, and regeneration is performed to receive the next sample derivatized product.

(15) Measurement/data processing The sample derivatized components separated and eluted by the separation column 12 sequentially flow into the flow cell 62 of the detector 61, detect the fluorescence intensity, and sequentially store the detected fluorescence in the memory of the data processing device 63. Calculate the concentration of each component. CR
The above calculation data and chromatogram are output to T66 and printer 65.

In addition, in order to improve the reliability of the analyzer, we have added a function to re-analyze the measurement sample when it is outside the preset standard value range (for example, 1710 or less or 10 times or more of the set standard value). have.

Note that the measurement of the standard sample is analyzed based on the flowchart shown in FIG. 5 above. In this case, the health of the reagent can be determined from the peak height 2 area of the chromatogram, and the health of the column and eluent can be determined from the shape of the peak (cracking, leading, tailing). If the above results are satisfactory, proceed to measurement of the unknown sample.

To diagnose the status of each unit during analysis, various sensors provided in each unit are monitored, and the status and messages of each unit are displayed on the CRT 66.

Table 6 shows an example of a CRT screen showing the status of each of the above devices. Furthermore, the analysis conditions shown in FIG. 6 are shown in Table 1. In addition,
The flow rates of the eluent and pretreatment liquid in this example were both 1 mΩ/min.

The CRT screen illustrated in FIG. 6 shows the analysis state of the 10th sample out of 30 samples.

The amount of pretreatment liquid, eluent, cleaning liquid, and waste liquid are measured by a weight sensor. Pretreatment liquid 71 and eluent 7 on the screen
2. The display of the liquid levels of the cleaning liquid 73 and waste liquid 74 changes according to the values measured by each weight sensor. below), and in the case of waste liquid, a certain amount or more (e.g.
(90% or more of the total), the liquid tank display on the corresponding screen flashes, and a comment such as ``Cleaning liquid shortage j'' is displayed in the message field 75.

The flow rate and pressure of pump 1 and pump 2 are also displayed at 76 (pump 1) and 77 (pump 2) on the CRT screen, and if there is an abnormality (for example, if the pressure is outside the set pressure range), the warming The same abnormality processing as in the upload processing is performed, and the message field 75 displays the device name shown in FIG.
Display messages such as stop time. The sample number being analyzed is displayed on the autosampler 78 on the CRT screen.

The temperatures of the sample cooling device 31, reaction vessel constant temperature bath 48, pre-column constant temperature bath 55, and separation column constant temperature bath 60 in the sample rack shown in FIG. 1 are also monitored by each temperature sensor and displayed on the CRT screen. If the measured temperature falls outside the preset temperature range, an abnormality process similar to the warm-up process is performed, and a message such as ``separation column thermostat temperature abnormality'' is displayed in the message column.

The state of the detector 61 is determined by monitoring the output light of the lamp, comparing it with the initial output light, and displaying, for example, the luminous intensity ratio on the detector 81 on the CRT screen. Then, when the output becomes less than a certain value (for example, 80% or less).

Detector 81 on the CRT screen flashes, message 111
175, a message such as [Please replace the lamp] is displayed.

Furthermore, each unit is equipped with a water leak sensor, which flashes the water leaking device on the CRT screen when water leaks.

Message aI75 will display "Water leak J, etc."

The analysis status is displayed on the analysis screen 82, including the current number of samples, remaining number of samples, elapsed time, required time, scheduled analysis end time, current time, noise level, drift value, etc. Make it easy to tell the time at a glance. When the analysis is completed, a message shown in FIG. 8 is displayed on the analysis screen 82.

Furthermore, to determine the life of the separation column or pre-column, it stores column pressure, number of samples measured, total usage time, measurement peak width, peak height, peak area, number of theoretical plates, retention time, etc.

It has a function to perform calculations, determine the lifespan of the column from these, and display it in the message 412175.

In addition, the lifetime of the column can be determined in the same manner from the measured peak height, peak width, peak area, number of theoretical plates, retention time, separation rate, etc. of the standard sample during calibration.

■ Data processing The data processing device 63 uses the stored data to create daily reports, group subjects by gender, age, region, medical condition, etc., create graphs showing changes in the same subject over time, etc. It has the function of performing arithmetic processing such as comparison with past data through pattern recognition using a neurocomputer that electrically reproduces the function of nerve cells.

It also has a function of creating a daily report through data processing and outputting it directly from the printer 65.

FIG. 9 shows a control system diagram of this embodiment.

The CPU 64 includes an autosampler 1, a pump 51゜57, a data processing device 63, a valve switching device 1-1, a sample cooling device 31 in the sample rack, a reaction vessel constant temperature bath 48,
Pre-column constant temperature bath 55, separation column constant temperature bath 60. The contact sensor 69 for sample counting, CR, T66, and printer 65 are controlled as programmed in advance, and various sensors including the empty sample testing detector 47 are monitored, and in the same manner as described above, depending on the situation. This process is performed using

FIG. 10 shows the operation panel.

On the operation panel 99 there are push buttons 100 for one analysis (
There are push buttons 101 for start) and data processing.

When performing an analysis, simply pressing the push button 100 automatically starts the analysis according to the procedure explained in FIGS. 2, 3, and 5.

In addition to the button, the start input can also be made by voice or handwriting. It can also be done using an IC card. In the case of data processing, when the push button 101 is pressed, various menus or messages appear on the screen of the CRT 66, and menu selections can be made using the input bezel 102. Furthermore, data editing is done by I, D, N.
o, can be used to search.

As explained above, in order to satisfy a so-called one-touch operation liquid chromatograph automatic analysis system that processes information from each component and various sensors at high speed, has a self-diagnosis function and a data processing function, it is necessary to have two internal memories. It is preferable to use a 32-bit CPU, which is a megabyte.

Especially for fast analysis, e.g. 1.0 mm I,D,X5
When using a separation column of .

FIG. 11 shows an example of an analysis program.

2. As a separation column to perform analysis within 8 minutes.
0mm1. D, X5. Omm (average particle size 2.5 μm silica 0DS) is used. As shown in the figure, it takes 1 minute from sample dispensing to derivatization reagent dispensing and mixing, 2.5 minutes for derivatization reaction, 1.5 minutes for concentration of sample derivative, and 3 minutes for separation and data processing, totaling 8 minutes. The analysis is programmed to be performed in

FIG. 12 shows an example of an analysis result obtained by performing analysis using the program shown in FIG. 11 using the analysis system of this embodiment shown in FIG.

The vertical axis shows fluorescence intensity and the horizontal axis shows retention time. The numerical value printed at the top of each peak is the retention time, i.e.
It shows the time from the start of analysis until each separated component reaches the detector. The measurement conditions were as shown in Table 1 above except for the separation column.

In the analysis, NE (norepinephrine), E (epinephrine), and DA (dopamine) are detected in the order, and finally the internal standard substance IP (isoproterenol) added by the internal standard solution is detected. The concentration of each component can be determined from each peak height or peak area.

This system is not limited to the analysis of catecholamines, but by partially changing the system configuration (reagents, columns, detectors, etc.), it can analyze amino acids, glycated hemoglobin, etc.
It can also be applied to the analysis of blood drugs such as prostaglandins or cyclosporin. For example, in the case of amino acids, the separation column is a cation exchange column and the detector is a spectrophotometer.

Similar measurements can be made by using ninhydrin as the derivatization reagent.

[Effects of the Invention] The liquid chromatography analysis system of the present invention uses a CPU to rapidly process information obtained by various sensors provided in each component device that constitutes the system, and at the same time, provides self-diagnosis functions and abnormality processing of each component device. Since it is equipped with self-management means for reagents and the like, it is possible to provide a so-called one-touch operation analysis system in which analysis is completely automated.

[Brief explanation of drawings]

Fig. 1 is a flow path system diagram of an embodiment of the present invention, Fig. 2 is a flow diagram of status diagnosis of each component device of the embodiment, Fig. 3 is a flow diagram of warm-up processing of the embodiment, and Fig. 4 is A side view of the autosampler of the example, Fig. 5 is a flowchart of the analysis of the example, Fig. 6 is a CRT screen diagram showing the status of each device during the analysis of the example, and Fig. 7 is a diagram at the time of abnormal stop of the example. C of
RT screen diagram, Figure 8 is a CRT screen diagram at the end of analysis of the example, Figure 9 is a control system diagram of the example, Figure 10 is a plan view of the operation panel of the example, and Figure 11 is the analysis of the example. FIG. 12 is a diagram showing a program, FIG. 12 is a chromatogram obtained in an example, and FIGS. 1-3 are a system diagram of a conventional analyzer. 1... Autosampler, 2... Sample container, 3, 8,
13゜17...Pump, 4...Nozzle, 5...Sample injector, 6...Sample loop, 7...
Precolumn, 9... Pretreatment liquid, 10, 15, 22.2
3... Valve, 11... Column switching valve, 12.
...Separation column. 14... Eluent, 16... Discharge pipe, 18... Reaction reagent, 19... Reaction coil, 20... Fluorometer,
21...Washing liquid, 31...Sample rack, 32.
... Sample stage, 35 ... Internal standard solution, 36
...Standard sample, 37...Reaction container, 38...
Nozzle cleaning tank, 39... Drain boat, 40...
Injection boat, 41... Drive mechanism, 43... Pipe, 44...
...Three-way valve, 45...Dispensing pump, 47...Empty sample detection detector, 48...Reaction container constant temperature bath, 49...Analysis department, 51.57...Pump, 55... - Pre-column constant temperature bath, 60... Separation column constant temperature bath, 61... Detector, 62... Flow cell, 63... Data processing device, 64... CPU, 65... Printer, 66...・
CRT, 71... Pretreatment liquid, 72... Eluent, 73
...Washing liquid, 74...Waste liquid, 75...Message column, 76...Pump 1.77...Pump 2.78.
・Autosampler, 79...Precolumn, 80...
Separation column. 81...Detector, 82...Analysis screen, 83-86.
・・Weight sensor, 87-98 ・Water leakage sensor, 99・
...Operation panel, 100, 101...Push button, 1
．． 02... Input pen. Fig. 1 (1 idiot) ＼, congratulations 64 ・ CPU 65 ... Printer 66 - CRT Fig. 31 ...... Sample rack 69, 7
0...Touch sensor Fig. Fig. Fig. Fig. Fig. 11 Fig. 12 Fig. O123 Holding time (minutes)

Claims (1)

[Claims] 1. A liquid chromatography analysis system for analyzing liquid components, which system includes an autosampler section, an analysis section, a control section, and various sensors, and the analysis section includes a precolumn, a separation column, a detector, and a control section. The various sensors are arranged in the operating parts of the autosampler section, the analysis section, and the control section, and the autosampler section includes a sample preparative distribution means, a reagent addition means, and a sample derivatization section. The precolumn has a function of adsorbing and concentrating the sample introduced from the autosampler section, and the separation column has a function of separating the derivatized sample concentrated by the precolumn and eluted into the eluent. The detector has means for measuring the concentration of each component of the derivatized sample separated by the separation column, and the data processing section is configured to perform arithmetic processing on the concentration measurement data;
It has arithmetic processing of the detection signals of each sensor, input/output of related data, and memory function, and the control section is configured to perform calculations based on a preset program and the calculation results of the data processing section.
A liquid chromatography analysis system characterized in that analysis is performed by controlling each of the above-mentioned means. 2. After starting the analysis, the analysis is performed based on the calculation results of the detection signals of the various sensors arranged in the operating parts of the autosampler part, analysis part, and control part, and the program set in advance in the control part. 2. The liquid chromatograph analysis system according to claim 1, wherein when an abnormality occurs, the analysis is interrupted and the details of the abnormality or the details of the abnormality and a solution are displayed on the CRT. 3. The liquid chromatograph analysis system according to claim 1 or 2, wherein the data processing section uses a CPU of 32 bits or more and an internal memory of 2 megabytes or more. 4. Analysis is started by inserting an IC card with the necessary analysis menu input, and the calculation results of the detection signals of the various sensors arranged in each operating section of the autosampler section, analysis section, and control section are calculated. 2. The liquid chromatograph analysis system according to claim 1, wherein the analysis is performed based on a program preset in the control unit and an analysis menu of the IC card. 5. The liquid chromatograph analysis system according to claim 4, wherein if an abnormality occurs during analysis, the analysis is interrupted and the details of the abnormality or the details of the abnormality and a solution are displayed on the CRT. 6. Warm up before starting sample analysis.
The liquid according to claim 1, characterized in that chromatographic noise and/or drift are measured at predetermined time intervals, and warming-up is continued until the noise and/or drift value falls within a predetermined value. Chromatographic analysis system. 7. A claim characterized in that, before sample analysis, the rate of change in retention time of a standard sample is measured, the calculated value is calculated from the measured value and the number of theoretical plates of the separation column, and the predicted life of the separation column is displayed on a CRT. The liquid chromatography analysis system according to item 1. 8. The liquid chromatograph analysis system according to claim 1, wherein the analysis time of all samples is calculated from the analysis time of one sample, and the analysis end time is displayed on the CRT. 9. The liquid chromatograph analysis system according to claim 1, further comprising means for self-diagnosing each part of the system before and during sample analysis, and means for displaying the diagnosis results on a CRT. 10. The liquid chromatograph analysis system according to claim 1, wherein the liquid chromatography analysis system is programmed to re-analyze the sample when the analysis value of the measurement sample is outside a preset value range.