JP3422092B2 - Liquid sample continuous measuring device and measuring method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is capable of rapidly processing and measuring a sample having minute solids, proteins, pollutants, etc., which are obstacles to analytical instruments, and particularly for online measurement of culture solution, fermentation solution and the like. The present invention relates to a suitable liquid sample continuous measuring device and measuring method.

[0002]

2. Description of the Related Art Conventionally, as a method for quantifying a substance to be detected in a sample, a fixed amount of the sample is injected into a continuous flow, and a spectrophotometer equipped with a flow cell, an atomic absorption spectrometer, or an electrochemical is provided. Known are a flow injection analysis method that leads to a detector and the like, and a flow analysis method that uses a bubble segment to separate the sample into and from the solution before and after sending the sample by segmenting the sample with bubbles at regular intervals.

These analysis methods have a short analysis time and can perform highly accurate analysis. In addition, the operations such as mixing, separation, chemical reaction, etc., which humans have performed so far in measurement, can be obtained in a continuous flow. There are advantages such as being able to. Especially, the flow injection analysis method requires less sample,
It does not require a complicated mechanism for strict insertion / removal of bubbles such as bubble segment, and has a feature that the device itself is small with a simple mechanism.

In recent years, analysis has been automated by combining an autosampler as an apparatus for automatically injecting a small amount of a sample into an analytical instrument by the flow injection analysis method. However, if the sample contains minute solids, proteins, pollutants, etc., such as culture broth and fermentation broth, it will be difficult to promote automation of analysis because various troubles will occur in the analytical equipment. There is.

[0005] For example, the sticking or contamination of solid matter in the piping of the equipment causes the piping to be clogged, causing abnormal high pressure. Even before these problems occur, for example, in the case of an auto sampler, a sample suction error, a weighing error, and a working wetted member may be damaged. Further, in the case of an analytical instrument, sensitivity fluctuations are caused by the attachment of contaminants to the detection section.

Particularly, in the case of an analytical instrument using a bio-related substance such as an enzyme, the attachment of proteins, microorganisms and the like can cause enzyme deactivation. Therefore, it is necessary to centrifuge or filter such a sample in advance before introducing the sample into the autosampler, which consumes a great deal of time and labor. Furthermore, when the sample exceeds the quantifiable concentration range of the analytical instrument, an operation of diluting the sample in advance is necessary. Specifically, it is an operation of collecting a fixed amount of each of the sample and the diluting solution, mixing and stirring them to obtain a solution having a uniform concentration, but this is also complicated and requires a long-time treatment. In addition, there is a problem that an error and a human error are added depending on a device to be used and a container. There is also known a diluting device in which the series of operations required for diluting is directly replaced by mechanical operation, but it has a drawback in that it has a complicated configuration and the length of time required for processing.

[0007] In particular, when performing so-called on-line measurement for continuously analyzing the concentration of a specific component in a solution such as a culture solution or a fermenting solution, which is continuously changing, obstacles relating to analytical equipment such as minute solids and proteins contained in the sample are eliminated. Of course, appropriate dilution must be carried out rapidly and accurate analysis must be possible. It is possible to separate minute solids and proteins from the sample solution using a dialysis membrane and supply only diffusible low molecular species to the analyzer. For example, there is known a method of measuring a permeated glucose amount by injecting a certain amount of blood with an injector and injecting it into a buffer solution flow, passing through a dialyzer so that proteins in blood do not enter the detector. (Japanese Patent Publication No. 61-2279). In this case, the partial permeation of glucose through the dialysis membrane also serves as a diluting effect.

However, in this method, since the dispersion pattern of the low molecular weight substance after permeation becomes large due to a slight change in the dialysis conditions such as the change in the rate of sending the sample, the change in the dialysis membrane becomes large. The reproducibility of the dialysis state is low, and there is a major drawback in terms of measurement accuracy. In addition, the reproducibility of the permeation state in the dialysis membrane is low because a small amount of the sample comes into immediate contact with the membrane of the dialyzer. Since the sample stock solution is supplied to the injector itself, it is difficult to avoid contamination of the injector. This also causes a problem that the weighing changes due to the attachment of solids and proteins in the injector, which is the weighing portion of the sample.

[0009]

DISCLOSURE OF THE INVENTION The present invention solves the above problems and eliminates minute solids, proteins, contaminants, etc. in a solution sample, which is an obstacle to analytical instruments, and makes it possible to quickly and rapidly determine components to be measured. It is an object of the present invention to provide a liquid sample continuous measuring device and a measuring method which can be accurately measured by an analytical instrument and which is particularly suitable for online measurement of a culture solution, a fermentation solution and the like.

[0010]

The liquid sample continuous measuring apparatus of the present invention comprises a filtration cell having a sample liquid passage and a carrier liquid passage which are in contact with each other through a semipermeable membrane, and a liquid to be measured containing a sample is contained in the filter cell. A measurement liquid feeding system including a measurement liquid feeding means for feeding a sample liquid passage at a constant flow rate, a carrier including a carrier feeding means for feeding a carrier at a constant flow rate into a carrier passage of a filtration cell A liquid feeding system, an injector to which the carrier liquid passage outlet of the filtration cell is connected to inject the carrier into the detector intermittently, a liquid feeding flow path of the buffer solution from the injector to the detector, and both liquid feeding means. After a lapse of a certain time after the start of liquid transfer, at least a part of the sample in the liquid to be measured in the filtration cell was stably transferred to the carrier through the semipermeable membrane to obtain the measurement target component.
The carrier containing is introduced into the injector and the detection is performed via the injector.
The control unit has a function of injecting the buffer solution into the detector through the flow path of the buffer solution to the container .

Further, it is preferable to provide a merging portion for connecting a diluent delivery system for delivering the diluent in the middle of the measurement solution delivery system. A merging portion is provided on the upstream side of the measurement liquid feeding means of the measurement liquid feeding system, and the merging portion has a sample liquid introduction port, a diluting liquid introduction port, and a discharge port, and one of the introduction ports is introduced. The other is an exclusive type valve unit that does not conduct flow passage to the outlet when the flow passage is connected to the outlet, and the control unit is configured to conduct the flow passage to the outlet at each time width for each inlet of the exclusive valve unit. It may further have a function of sequentially switching to and repeatedly controlling. The merging portion may be provided on the downstream side of the measuring liquid feeding means of the measuring liquid feeding system, and the diluting liquid feeding system may include a diluting liquid feeding means for feeding the diluting liquid to the merging portion at a constant flow rate. good.

The liquid sample continuous measuring method of the present invention uses a measuring liquid delivery system connected to a sample passing passage of a filtration cell having a sample passing passage and a carrier passing passage which are in contact with each other through a semipermeable membrane. A step of sending a measured liquid containing at a constant flow rate, a step of sending a carrier at a constant flow rate by a carrier sending system connected to a carrier flow path of the filtration cell, a measured solution and a sending of carrier After a certain time has passed since the start,
By the injector to which the carrier flow passage outlet of the filtration cell is connected, at least a part of the sample in the liquid to be measured in the filtration cell is stably transferred into the carrier through the semipermeable membrane to obtain a carrier. Via the flow path of the buffer solution to the detector
And injecting it into the detector.

[0013]

In the measurement liquid delivery system, the measurement liquid containing the sample is delivered to the sample passage of the filtration cell at a constant flow rate by the measurement liquid delivery means. Similarly, in the carrier liquid feeding system, the carrier is fed to the carrier liquid feeding passage at a constant flow rate by the carrier liquid feeding means. In the filtration cell, at least a part of the sample in the liquid to be measured flowing through the sample liquid passage moves into the carrier flowing through the carrier liquid passage through the semipermeable membrane.

Since the liquid to be measured and the carrier are respectively sent at a constant flow rate, this movement can be carried out sufficiently stably after a certain period of time. The control unit injects the carrier obtained by the stable movement in this way into the detector at an appropriate time, that is, after a certain period of time elapses from the start of the liquid feeding of both liquid feeding means. In other words, the movement of the sample in the filtration cell is performed between the liquid to be measured having a constant concentration and being fed at a constant flow rate and the carrier being fed at a constant flow rate, so that the solution feeding time is stable to some extent. Can be obtained. That is, since a sufficient amount of the sample can be controlled so as to be in contact with the semipermeable membrane for as long as necessary until it is constantly stabilized, the reproducibility of the moving state in the semipermeable membrane is enhanced. After all, in the present invention, unlike the case where the segmented sample is momentarily brought into contact with the semipermeable membrane, the sample permeates the measurement target component in a state where the sample is filled in the sample passage of the filtration cell, The state of permeation into the carrier flow path is averaged, enabling highly reproducible measurement. Furthermore, since the injector is not connected to the sample passage side, the injector is liable to be contaminated and is less likely to be contaminated.

In the filtration cell, at least a part of the sample in the liquid to be measured flowing through the sample liquid passage moves into the carrier flowing through the carrier liquid passage through the semipermeable membrane. In this way, the carrier containing the sample is injected into the detector by the injector. In detail, this means that in the injector the carrier is injected into the carrier for the detector and is measured by the detector connected after the injector. In this way, the measurement process of one sample is completed.

The flow direction of the sample and the flow direction of the carrier are
Allow parallel flow on both sides of the membrane in the same or opposite directions. The semipermeable membrane referred to in the present invention is a membrane that allows some components in a solution or dispersion system to pass through but does not allow other components to pass through. For example, among substances contained in a sample, a regenerated cellulose-based membrane, a polysulfone-based membrane as a membrane that does not pass macromolecules such as minute solids and proteins, which are inconvenient to the detector, and can pass a measurement target component that is a low molecule, Examples thereof include a fluororesin film and a carbonate film.

A structure having a sample liquid passage and a carrier liquid passage that are in parallel contact with each other partially via a semipermeable membrane is a filtration cell. The filtration cell is formed of a pair of two blocks each having a groove in a mirror-symmetrical position, and both ends of the groove of each block are penetrated so as to form a flow path toward the entrance and exit of the cell provided outside the block. ing. Then, the two blocks are aligned such that the grooves face each other, and the semipermeable membrane is sandwiched in the portions where the grooves face each other. The sample flow direction and the carrier flow direction are set to flow in the same direction or in opposite directions in parallel on both sides of the membrane. By increasing the effective area (for example, the product of the length in the flow direction and the width in the direction parallel to the membrane in the direction perpendicular to the flow direction) of the groove of the filtration cell, the integrated amount of the component that permeates the carrier increases. Also, if this effective area is large, it is possible to obtain a uniform effect of filtration that alleviates fluctuation factors due to pump pulsation, etc., but if it is too large, when switching the sample or standard solution,
The time required for replacement becomes long. Therefore, the size of this effective area is preferably set to a size that matches the detection concentration range of the detector in the subsequent stage.

Particularly, regarding the width of the groove, if the width is too wide, the film surface of the semipermeable membrane fluctuates, and if it is too narrow, minute solids contained in the sample are clogged, so that these phenomena must be set within a range that does not occur. However, it is preferably within the range of 0.5 to 5 mm. Further, the depth of the groove is preferably about the same as the width, but the depths of the sample side and the carrier side may be different, and the shape of the cross section of the groove with the liquid flowing direction as a normal line is also rectangular. The shape can be a half circle, a half ellipse, or the like. In addition, the flow channel shape in the flow direction of the groove can be various shapes such as a linear shape, a spiral shape, and a bent shape, but the liquid flow is partially retained or the flow channel is partially narrowed. It is desirable that the shape is such that there is no

In the present invention, the sample introduced to the sample passage is
It also contains the actual sample solution and standard solution when the detector requires calibration. Therefore, in order to calibrate the detection device, a valve is arranged as a switching means for sequentially switching a plurality of sample solutions and standard solutions. As this valve, a 3-way valve having a 3-way switching function is often used. Further, a plurality of three-way valves are arranged and used so that a plurality of standard solutions can be supplied.

In addition to the standard solution used for calibrating the sample and the detector, it is also possible to dispose distilled water or the like as a cleaning solution in order to clean the filtration cell and the pipes before and after it. A sample liquid transport pump that serves as a measurement liquid delivery means for delivering a sample liquid through a sample passage, and a carrier delivery pump that serves as a carrier delivery means for passing a carrier through a carrier passage of the filtration cell. As the above, various conventionally known pumps such as a plunger type pump, a geared pump, and a peristaltic pump can be used.

The sample liquid transfer pump is preferably a pump having resistance to minute solids and adhered substances, and it is more preferable to use a peristaltic pump. The position of the sample solution transfer pump can be located at the sample supply side or the discharge side of the sample passage of the filtration cell.However, when the sample solution transfer pump is located at the former position, the effect of further stirring the sample and diluent is increased. Is desirable and that the inside of the sample passage of the filtration cell is not depressurized to generate bubbles.

Also in the carrier transfer pump, it can be arranged on the carrier supply side to the filtration cell or on the discharge side of the carrier flow path of the filtration cell, but like the sample solution transfer pump, the carrier flow path of the filtration cell. It is desirable to place it in the former position so as not to generate bubbles inside. Each pump used in the present invention has a mechanism capable of delivering one liquid, and is a peristaltic pump capable of simultaneously operating multiple tubes with rollers rotating coaxially with one power source. Is, of course, equivalent to multiple pumps in one unit.

The passing speed of the carrier through the carrier passage is
It is possible to set how much carrier amount the component to be measured that is transmitted per unit time is received. That is, the magnitude of dilution in the filtration cell can be varied. For example, the higher the liquid passing speed of the carrier, the higher the dilution ratio, and conversely, the slower the carrier flowing speed, the lower the dilution ratio. It is adjustable.

In view of these characteristics, it is desirable that the carrier transfer pump should be a pump excellent in stability and accuracy of liquid transfer in order to maintain a stable dilution rate in the filtration cell. As the carrier in the present invention, for example, distilled water, various buffers, etc. can be used, but it is also possible to add reagents and the like required for detection and use them.

In the present invention, the carrier containing the analyte of the sample discharged from the carrier passage of the filtration cell is sent as a sample to an injector having a detector connected to the latter stage, and is detected by the detector. I have to. The injector is a device that weighs a fixed amount of sample and injects it into a continuous flow to the detector, and uses a mechanism such as a 6-way switching valve that is generally adopted in auto samplers and the like. It is a device.

With the above configuration, only when the concentration of the component to be measured in the sample is in a relatively low and limited range,
The ratio of transmission through the semipermeable membrane (hereinafter referred to as the transmittance) can be kept substantially constant. It has been found that when the density exceeds the range and enters a relatively high density region, the transmittance decreases. Therefore, further investigation was conducted on a configuration that enables accurate measurement even for a sample having a relatively high concentration of the measurement target component.

If the relationship between the sample concentration and the higher-order curve based on multiple points of the sample concentration on the carrier side containing the permeated sample is determined using standard solutions having a plurality of concentrations, the components to be measured in the sample up to the high concentration region are obtained. Even when quantifying the concentration of, even in the case of online measurement where high-speed measurement is required up to quantification, it takes a long time to calibrate by measuring a large number of standard solutions in advance and preparing multiple standard solutions. There is a drawback that it becomes complicated.

As a liquid sample continuous measuring device having no such drawbacks, a confluence portion for connecting a diluting liquid feeding system was further provided in the middle of the measuring liquid feeding system. With such a configuration, it is possible to perform dilution by adding a diluting liquid to the sample before bringing the semipermeable membrane and the sample into contact with each other, and supplying a treatment liquid in proportion to the concentration of the measurement target component of the original sample for measurement. It will be possible. That is,
Even if the concentration of the measurement target component is in a wide range from low concentration to high concentration, supply and measure the treatment liquid obtained by transmitting the measurement target component in proportion to the concentration of the measurement target component of the original sample. Is possible.

As one form of the merging portion as described above, there is one in which the merging portion is provided on the upstream side of the measuring liquid feeding means of the measuring liquid feeding system. In this case, the confluent section has a sample liquid inlet, a diluent inlet, and an outlet, and when one of the inlets is in flow passage with the outlet, the other is not in exclusive connection with the outlet. Further, the control unit is further provided with a function of sequentially switching and repeatedly controlling each introduction port in the exclusive valve unit so as to conduct the flow path to the outlet port in each time width.

The exclusive valve section has two inlets, a sample liquid inlet and a diluent inlet, and one outlet.
When one inlet is in flow passage with the outlet, the other inlet may be formed by a three-way valve or a plurality of on-off valves or a combination thereof, which is not in flow passage with the outlet. In addition, the term “exclusive” as used in the present invention means that two or more inlets that can be in fluid communication with the outlet are not selected at the same time.

The valve drive system may be an electromagnetic system, an air pressure system or the like. The electromagnetic system is preferable because of its short response time, opening and closing accuracy, and small power source, and a diaphragm valve is usually used. An electromagnetic valve is used.

The control unit controls the passage time of each inlet in the exclusive valve unit, the sequential switching, the repetition, and the like. The shorter the cycle for switching the inlet of the exclusive valve section, the more uniform the mixing can be.
If the response speed of the valve is lower than the response speed, mixing cannot be performed in an accurate ratio. In the case of an electromagnetic valve, since the operating time is generally about 10 msec, the flow passage conduction switching period is basically controlled in the range of about 50 msec to 10 sec, and the flow passage conduction time is controlled in the range of about 50 msec to 10 sec. .

The flow channel conduction time is set to 0 msec, and the flow channel conduction time ratio is set to 100: 0 or 0: 100.
It is also possible to selectively and continuously supply only one of the sample liquid and the diluting liquid. For example, in a system in which it is necessary to clean the inside of the pipe and the inside of the sample storage container, the clean diluent side is continuously connected for a certain period of time, and the sample transfer from the sample tank to the exclusive valve part is performed quickly. in case of,
The sample is controlled so as to be continuously connected to the flow channel for a certain period of time.

In this structure, an opening / closing valve can be arranged in each pipe so that all the flow paths are closed when the apparatus is stopped. Further, when the exclusive-type valve section is composed of a plurality of on-off valves, it is possible to close all the inlets so that the inlets are not electrically connected to the outlets.

The sample introduced to the sample introduction port of the exclusive type valve section includes the actual sample solution and the standard solution when the analytical instrument requires calibration. Therefore, in the previous stage of the sample solution inlet, a three-way switching device similar to the exclusive type valve section that switches between the sample solution and the standard solution for calibrating the detection device can be arranged, and a plurality of standard solutions can be supplied. A plurality of three-way switching devices can be arranged as much as possible.

In the exclusive valve section, the two inlets are time-divided into the outlets in a flow-wise manner so that the sample liquid and the diluting liquid are mixed, and the sample liquid and the diluting liquid are mixed by this single pump. Since it is inhaled by, it is possible to form an accurate mixed liquid of two liquids.

As another form of the merging portion as described above, the merging portion is provided on the downstream side of the measuring liquid feeding means of the measuring liquid feeding system. In this case, the diluent delivery system is an apparatus further provided with a diluent delivery pump as a diluent delivery means for delivering the diluent at a constant flow rate to the junction. In this case, the pump used for the sample liquid transfer pump described above can be appropriately used as the diluent transfer pump.

The confluence ratio of the measurement liquid containing the sample and the diluting liquid, that is, the dilution ratio before being introduced into the filtration cell, can be easily adjusted by the flow rate ratio between the two pumps of the sample liquid conveying pump and the diluting liquid conveying pump. it can. Further, since the total flow rate is increased due to the confluence of the diluting liquid, the sample transport speed is naturally increased, so that the replacement of the sample in the filtration cell is accelerated, and there is an advantage that contamination and clogging in the filtration cell are less likely to occur.

[0039]

EXAMPLES The configuration of the liquid sample continuous measuring apparatus of the present invention will be described in detail below with reference to the examples shown in the drawings. In addition,
What is simply expressed as% means% by weight. Example 1 FIG. 1 shows a liquid sample continuous measuring apparatus of the present invention,
It is a systematic diagram of the 1st example applied to the flow injection analyzer for measuring the glucose concentration in a solution.

The sample solution (14), the standard solution (15) and the cleaning solution (3) are three-way electromagnetic valves (24) and (25).
Then, any one of the liquids is selected and supplied to the sample liquid passage (5) of the filtration cell (8) by the sample liquid transfer pump (9). The measurement liquid delivery system is configured as described above. Also,
The carrier (13) is supplied to the carrier liquid passage (6) of the filtration cell (8) by the carrier transfer pump (10). The carrier liquid feeding system is configured as described above.

The filtration cell (8) has a sample liquid passage (5) and a carrier liquid passage (6), which are in contact with each other in parallel through a semipermeable membrane partially inside, and is an object of measurement of low molecular weight substances. A part of the component permeates the sample liquid passage (5) to the carrier liquid passage (6) and is carried to the sample injection part (20) composed of an injector, where 5 μl is weighed and the detector ( 21).

Explaining this step in detail, the sample liquid (14), which is the liquid to be measured containing the sample, is the sample liquid passage (5) of the filtration cell (8), which is the measuring liquid sending means at a constant flow rate. The liquid is transferred by the liquid transfer pump (9). Similarly, the carrier (13) is sent to the carrier passage (6) at a constant flow rate by the carrier transfer pump (10) which is a carrier sending means. In this state, in the filtration cell (8), at least a part of the sample in the liquid to be measured flowing through the sample liquid passage (5) is a semipermeable membrane which is a semipermeable membrane in the carrier flowing through the carrier liquid passage (6). Move through (7).

Since the liquid to be measured and the carrier are respectively sent at a constant flow rate, this movement can be carried out sufficiently stably after a certain period of time. The control unit (11) buffers the carrier obtained by such stable movement at an appropriate time, that is, after a certain period of time elapses after the start of liquid feeding of both pumps, by the sample injection unit (20). Inject into liquid. The injected sample is measured by the detector (21) connected after the sample injection part (20). Such a certain period of time is set in advance according to each case by testing the device under various conditions (flow rate of sample and carrier, concentration, piping, etc.). In other words, the movement of the sample in the filtration cell is performed between the liquid to be measured having a constant concentration and being fed at a constant flow rate and the carrier being fed at a constant flow rate, so that the solution feeding time is stable to some extent. Can be obtained. That is, since a sufficient amount of the sample can be controlled so as to be in contact with the semipermeable membrane for as long as necessary until it is constantly stabilized, the reproducibility of the moving state in the semipermeable membrane is enhanced. Transport of the sample from the sample injection part (20) to the detector (21)
Buffer solution (1) for detector by buffer solution transfer pump (19)
8), and the detected liquid is the waste liquid bottle (2
It is discharged to 2). In this way, the control unit (11) controls the operations of the pumps (9) (10) (11), the valves (24) (25) and the sample injection unit (20), and is a microcomputer and a necessary input / output device. It is composed of peripheral equipment such as.

The sample solution transfer pump (9) and the carrier transfer pump (10) were both leaky peristaltic pumps, and the flow rates were 1.0 ml / min and 0.75, respectively.
It was set to ml / min. A plunger type pump was used as the buffer solution transfer pump (19), and the flow rate was 1.0 ml / m.
in. In the filtration cell (8), the groove of each of the two blocks forming the carrier liquid passage (6) and the sample liquid passage (5) on both sides of the semipermeable membrane (7) has a depth of 0.5 mm, A rectangular cross section with a width of 2 mm, a length of 40 mm, and a semipermeable membrane with a molecular weight cutoff of 300,000, Spectra / Pore CE type (Spectrum Medica)
l Industries, Inc.) dialysis membrane was used.

The buffer was 100 mM sodium phosphate buffer (pH 6), and distilled water was used as the carrier (13) and the washing solution (3). The detector (21) is 37
A flow cell is placed inside a constant temperature bath maintained at ℃. Further, an immobilized enzyme electrode for glucose detection and an Ag / AgCl reference electrode are arranged in this flow cell,
Further, the counter electrode, which is in contact with the liquid in the flow cell and is made of a stainless steel connection joint, is adjacent to the counter electrode. It should be noted that potentiostat was added to the immobilized enzyme electrode and to the Ag / AgCl reference electrode +
A voltage of 0.6 V is applied, an output current value is obtained based on the glucose amount of the injected sample, and the peak height of the waveform of the current value is detected as a response value.

The method for producing the above-mentioned immobilized enzyme electrode for detecting glucose will be described below. The side surface of a platinum wire with a diameter of 2 mm is coated with heat-shrinkable Teflon, and one end of the wire is rasp and
Smooth it with # 500 emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode (hereinafter abbreviated as SCE) as a reference electrode, electrolytic treatment is performed in 0.1 M sulfuric acid at +2.0 V for 5 minutes. After that, the platinum wire was washed well with water, dried at 40 ° C. for 10 minutes, and 10% γ-
It was immersed in an anhydrous toluene solution of aminopropyltriethoxysilane for 1 hour and then washed. The enzyme was immobilized on the aminosilanized platinum wire as follows.

Glucose oxidase (Sigma II, type II) 5 mg and bovine serum albumin (Sigma, Fraction V) 5 mg were dissolved in 100 ml of sodium phosphate buffer (pH 7) 100 mM, and glutaraldehyde was added to 0.2%. To be added. 5 μl of this mixed solution was quickly put on a platinum wire prepared in advance, dried and cured at 40 ° C. for 15 minutes, and then stored in 100 mM sodium phosphate buffer (pH 6). [Effects of the first embodiment] The filtration cell can eliminate minute solids, proteins, contaminants and the like in the solution sample, and analyze quickly and accurately.

[Embodiment 2] FIG. 2 is a system diagram of a second embodiment in which the liquid sample continuous measuring apparatus of the present invention is applied to a flow injection analyzer for measuring the glucose concentration in a solution. In the drawings, the members indicated by the same reference numerals as those of the first embodiment are the same as those used in the first embodiment, and therefore the description thereof will be omitted.

In the present embodiment, the exclusive type valve section (4) which is composed of a three-way electromagnetic valve which serves as a confluent section for connecting the measuring solution sending system and the diluting solution sending system described later is large. It is a feature. Diluent (1
2) is connected to the diluent inlet (2). Sample (1
4) or the standard solution (15) is connected to the sample solution inlet (1). That is, in the preceding stage of the sample solution inlet (1), the exclusive valve part (so that the standard solution (15) is connected when calibrating the analytical instrument and the sample (14) when quantifying is performed. A sample switching valve (17) similar to 4) is arranged. The sample (14), the standard solution (15), the sample switching valve (17) and the sample solution transport pump (9) constitute a measurement solution delivery system.

Sample liquid transfer pump (9) for feeding liquid from the outlet (3) of the exclusive valve section (4) to the filtration cell (8).
Is aspirated at a flow rate of 1 ml / min using a peristaltic pump, and liquid is fed. The control unit (11) performs the substantial drive and control of the exclusive valve unit (4) in addition to the control function described in the first embodiment.
ON the drive voltage application in the exclusive valve section (4),
It is turned off, and the respective inlets are sequentially switched to the outlet (3) in the individual time width so that the flow passage 9 is conducted, and the repetitive control is performed.

When the three-way electromagnetic valve is turned on, the sample liquid inlet (1) and the outlet (3) are connected to each other, and when the three-way electromagnetic valve is turned off, the diluent inlet (2) and the outlet (3) are connected to each other.
FF → ON → OFF is repeated and the switching cycle is set to 1
Seconds and an ON: OFF time ratio of 1: 3 were used to obtain a mixing ratio corresponding to a time width ratio. When the sample solution and diluent are connected to their respective inlets, the sample liquid inlet (1)
And flow passage conduction between the outlet (3) and the flow passage between the diluent inlet (2) and the outlet (3) are set, and a predetermined mixing ratio is obtained by the ratio.

In the inside of the filtration cell (8), the carrier liquid passage (6) and the sample liquid passage (5) are partially opposed to each other with the semipermeable membrane (7) interposed therebetween, and the carrier transfer pump (10 '). ), Using a plunger type pump, flow rate 0.75 ml / m
In, the carrier (13) is sent to the carrier liquid passage (6), receives the component to be measured transmitted from the sample liquid passage (5) through the semipermeable membrane (7), and receives the sample injection part (20). Is sent to. The liquid flow through the sample liquid passage (5) is stored in the waste liquid bottle (16).

In the sample injection part (20), 5 μl is weighed and injected into the flow of the buffer solution (18) from the bottle to the detector (21) by the pump (19). The buffer solution (18) was 100 mM sodium phosphate buffer solution (pH
6) is used to dilute (12) and carrier (13)
For this, distilled water was used.

The results of measuring response values by sequentially using glucose standard solutions up to 1000 mM as the standard solution (15) are shown in FIG. 3B. For comparison, the diluent (1
2) and the exclusive valve part (4) are omitted and the switching valve part (17) is directly connected to the sample liquid transfer pump (9), which corresponds to the structure of the first embodiment. The result of measurement using the same standard solution is shown in FIG.
Of A.

For reference, FIG. 4 shows the response values when the low concentration standard solution was directly injected into the sample injection part (20). As shown in FIG. 4, when a glucose solution equivalent to 500 nA is measured, the relationship between the concentration and its response value is linear.

However, as shown in FIG. 3A, when a high-concentration glucose solution is directly passed through the filtration cell (8), when the original glucose solution concentration exceeds a certain concentration, a part of glucose is The response value of the permeated carrier was not proportional to the glucose concentration. That is, the transmittance was lowered.

As in the liquid sample continuous measuring apparatus of the second embodiment, a high concentration sample is diluted with the exclusive valve section (4) and then the sample passage (5) of the filtration cell (8) is used. )
As shown in B of FIG. 3, the linearity between the response value and the concentration of the measurement target component contained in the solution can be obtained by passing the solution through. That is, as can be seen from FIG. 3A, in the configuration of Example 1, linearity is obtained in a relatively low concentration range of glucose concentration of 200 mM or less, but it becomes non-linear at a concentration higher than that. As described above, in the configuration of the first embodiment, the concentration range of the measurement target is limited to a relatively low concentration, whereas in the configuration of the second embodiment, the measurement target of a higher concentration can be measured. It can be seen from B in FIG. . Similarly, yogurt was passed through as a sample to measure the amount of glucose, but no decrease in sensitivity was observed for 24 hours, and reproducible results were obtained.

[Effect of the Second Embodiment] By removing fine solids, proteins, contaminants, etc. in the solution sample with the filtration cell, and mixing and diluting in the previous stage of the filtration cell, a wide concentration range can be obtained. The measurement target component can be analyzed quickly and accurately. Moreover, since the exclusive valve section distributes the two inlets to the outlets in a flow-wise manner and conducts them, and also sucks in with a single pump, stable and accurate dilution of the sample becomes possible, and analysis is performed. The mixing / dilution ratio can be easily set according to the measurement sensitivity of the device.

[Embodiment 3] FIG. 5 is a system diagram of a third embodiment in which the liquid sample continuous measuring apparatus of the present invention is applied to a flow injection analyzer for measuring the glucose concentration in a solution. In the figure, the members indicated by the same reference numerals as those of the first and second embodiments are the same as those used in the first and second embodiments, and therefore the description thereof will be omitted.

The major feature of this embodiment is that it has a confluence section (4 ') for connecting the measurement liquid delivery system and the diluent delivery system. The merging portion (4 ') is simply a T-shaped tube. Sample solution (14), standard solution (1
5) and the washing solution (3) are supplied to the confluence section (4 ′) by the sample solution transfer pump (9) by selecting one of the three solutions by the electromagnetic valves (1) and (2). The above is the measurement liquid delivery system. The diluent (12) sent by the diluent transfer pump (11) is also supplied to the merging section (4 ′). The above is the diluent delivery system. A sample diluted by joining at the joining section (4 ′) is supplied to the sample passage (5) of the filtration cell (8), and the carrier (13).
Is a carrier transfer pump (10) for filtration cell (8)
Is supplied to the carrier liquid passage (6).

The filtration cell (8) has a sample liquid passage (5) and a carrier liquid passage (6) which are in contact with each other in parallel via a semipermeable membrane inside, and is an object of measurement of low-molecular weight substances. Part of the components permeate from the sample liquid passage (5) through the semipermeable membrane (7) to the carrier liquid passage (6), and are carried to the sample injection part (20), where a fixed amount of the detector ( 21). The sample is transferred to the filtration cell and the detector (21) by the buffer transfer pump (1
9) and the detected liquid is discharged to the waste liquid bottle (22).

A peristaltic pump was used for each of the diluent transport pump (23), the sample solution transport pump (9), and the carrier transport pump, and the flow rate was 3.5 ml / m.
in, 1.0 ml / min, and 1.0 ml / min. A plunger pump was used as the buffer solution transport pump (19), and the flow rate was 1.0 ml / min.

The filtration cell (8) is almost the same as that used in Example 1, except that the inner length of the groove shape of each of the two blocks forming the sample passage (5) is 120 mm.
The same configuration was used except that. The buffer was 100 mM sodium phosphate buffer (pH 6), and the same carrier (13) was used. Distilled water was used as the diluting solution (12) and the washing solution (3).

The results of measuring the response values by sequentially using glucose standard solutions up to 1000 mM as standard solutions are shown in FIG.
B of FIG. Further, for comparison, when the sample is directly supplied to the filtration cell (8) without joining the diluting liquid (12), this also substantially corresponds to the configuration of Example 1, but a similar standard is used for this configuration. The result measured using the liquid is shown in Fig. 6A.
Shown in.

FIG. 7 shows the response values when the low concentration standard solution was directly injected into the sample injection part (20). As shown in FIG. 7, when a glucose solution equivalent to 500 nA is measured, the relationship between the concentration and its response value is linear.
However, as shown in FIG. 6A, when a high-concentration glucose solution is directly passed through the filtration cell (8), when the original glucose solution concentration exceeds a certain concentration, some of the glucose permeates the carrier. Response value was not proportional to glucose concentration, that is, the transmittance was decreased.

As in the liquid sample continuous measuring apparatus of the third embodiment, after diluting a high-concentration sample by confluence of the diluting liquid, the sample is passed through the sample passage of the filtration cell (8). As shown in FIG. 6B, the linearity between the response value and the concentration of the measurement target component contained in the solution can be obtained up to the high concentration region. Similarly, yogurt was passed through as a sample to measure the amount of glucose, but no decrease in sensitivity was observed for 24 hours, and good reproducible results were obtained.

[Effects of the Third Embodiment] A filter cell is used to eliminate minute solids, proteins, contaminants, etc. in the solution sample,
Moreover, by mixing and diluting in the previous stage of the filtration cell,
It becomes possible to analyze the measurement target component in a wide concentration range quickly and accurately. Further, since the sample transport speed increases with the dilution due to the confluence of the diluting liquid, the replacement of the sample in the filtration cell becomes faster, the analysis time can be shortened, and contamination and clogging in the filtration cell are less likely to occur.

[0068]

EFFECTS OF THE INVENTION Micro-solids, proteins in solution samples,
It eliminates contaminants, etc., and can quickly and accurately measure a wide range of components to be measured with analytical instruments.

[Brief description of drawings]

FIG. 1 is a system diagram of a first embodiment in which the liquid sample continuous measurement apparatus of the present invention is applied to a flow injection analysis apparatus for measuring glucose concentration in a solution.

FIG. 2 is a system diagram of a second embodiment in which the liquid sample continuous measurement apparatus of the present invention is applied to a flow injection analysis apparatus for measuring glucose concentration in a solution.

FIG. 3 is a graph showing the relationship between the original glucose concentration and the response value obtained by measuring the response value by sequentially using the high-concentration standard solutions of glucose by the liquid sample continuous measuring devices of the first and second embodiments. The graph showing.

FIG. 4 is a graph showing a relationship between a glucose concentration and a response value when a low-concentration glucose standard solution having a known concentration is directly injected from a sample injection part in the liquid sample continuous measurement apparatus of the second embodiment.

FIG. 5 is a system diagram of a third embodiment in which the liquid sample continuous measurement apparatus of the present invention is applied to a flow injection analysis apparatus for measuring glucose concentration in a solution.

FIG. 6 is a graph showing the relationship between the original glucose concentration and the response value obtained by sequentially using the high-concentration standard solutions of glucose by the liquid sample continuous measuring apparatus of the third embodiment to measure the response value. . In FIG. 6, B is a filtration cell (8), omitting the confluence part (4) with the diluting liquid (12), and directly carrying out the sample passage part (5) of the filtration cell (8) without dilution. ) Shows the measurement results when the solution on the sample side was passed through.

FIG. 7 is a graph showing a relationship between a glucose concentration and a response value when a low-concentration glucose standard solution having a known concentration is directly injected from a sample injection part in the liquid sample continuous measurement apparatus of the third embodiment.

[Explanation of symbols]

4, 4'merging section 5 Sample passage 6 Carrier flow path 7 Semi-permeable membrane 8 filtration cells 9 Sample liquid transfer pump 10, 10 'carrier transfer pump 11 Control unit 12 Diluent 13 career 14 samples 15 standard solution 18 buffer 19 Buffer transfer pump 20 Sample injection part 21 detector 23 Diluent carrier pump

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-1-94263 (JP, A) JP-A-5-302871 (JP, A) JP-A-4-84771 (JP, A) JP-A-5- 333028 (JP, A) Japanese Patent Publication Sho 61-20279 (JP, B1) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 35/06-35/08 C12Q 1/00-1/70 G01N 1/00-1/44

Claims (5)

(57) [Claims] 1. A filtration cell having a sample liquid passage and a carrier liquid passage which are in contact with each other through a semipermeable membrane, and a measurement liquid for feeding a liquid to be measured containing a sample to the sample liquid passage of the filtration cell at a constant flow rate. The measurement liquid delivery system including the liquid delivery means, the carrier delivery system including the carrier delivery means for delivering the carrier at a constant flow rate into the carrier passage of the filtration cell, and the carrier passage outlet of the filtration cell are connected An injector for intermittently injecting the carrier into the detector, a flow path for sending the buffer solution from the injector to the detector, and a certain time after the start of the solution sending of both solution sending means, in the filtration cell At least a part of the sample in the liquid to be measured is stably transferred to the carrier through the semipermeable membrane ,
The carrier containing is introduced into the injector and the detection is performed via the injector.
A liquid sample continuous measuring device comprising a controller having a function of injecting a buffer solution into a detector by a flow path of a buffer solution to the container . 2. The liquid sample continuous measuring apparatus according to claim 1, wherein a merging portion for connecting a diluent delivery system for delivering the diluent is provided in the middle of the measurement solution delivery system. 3. The merging portion is provided on the upstream side of the measuring liquid feeding means of the measuring liquid feeding system, and the merging portion has a sample liquid introducing port, a diluting liquid introducing port, and an outlet, and When one of the ports is in flow passage communication with the outlet, the other is an exclusive valve unit that does not flow in communication with the outlet, and the control unit has outlet ports with individual time widths for each inlet of the exclusive valve unit. 3. The liquid sample continuous measuring apparatus according to claim 2, further comprising a function of sequentially switching and repeatedly controlling so that the flow path is electrically connected to. 4. A diluting liquid delivery system in which the merging portion is provided on the downstream side of the measurement liquid delivery means of the measurement fluid delivery system, and the diluting fluid delivery system delivers the diluting liquid to the merging portion at a constant flow rate. The liquid sample continuous measuring apparatus according to claim 2, further comprising means. 5. A solution to be measured containing a sample is kept at a constant flow rate by a measurement solution delivery system connected to a sample passage of a filtration cell having a sample passage and a carrier passage which are in contact with each other through a semipermeable membrane. In the step of sending the carrier at a constant flow rate by the carrier sending system connected to the carrier passage of the filtration cell, after a certain time has elapsed after starting the sending of the solution to be measured and the carrier The carrier obtained by stably transferring at least a part of the sample in the liquid to be measured in the filtration cell into the carrier through the semipermeable membrane by the injector to which the carrier passage outlet of the filtration cell is connected. Buffer to the detector
A method for continuously measuring a liquid sample, which comprises a step of injecting a liquid into a detector through a liquid supply channel .