JPH09126961A - Concentration measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to perform the highly accurate measurement of a sample, which is liable to be contaminated, by a constitution, wherein a specified opening and closing mechanism is provided in a flow path, the opening is performed at a constant time interval for obtaining a specified liquid sending amount and the dissolved transferred quantity is detected by the specified procedure. SOLUTION: A pump 3 is driven, and liquid such as buffer liquid is made to flow through the lower-side flow path of a separating mechanism 9. An on/off valve 4 is opened during the time, wherein sample liquid to be measured flows through the upper-side flow path of the separating mechanism 9 by a specified amount, five times or more of the volume of the flow path. The output of a detector 11 during the time period is read out. In this output reading, data collection is started with the time point when the contact of the sample and a separating film 10 is started as a starting point. After the contact with the film 10 is finished, the data to the point where the output decreases are stored, and the maximum point and the minimum point of the output are obtained. Furthermore, S/N can be also improved by averaging the outputs in the vicinities of the maximum and minimum points.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention prevents contamination of a detector when analyzing a liquid sample, especially a fermented liquor, can reduce the sample consumption without being affected by turbidity, and is highly accurate and easy to maintain. The present invention relates to an analyzer.

[0002]

2. Description of the Related Art In a conventional concentration measuring device, a sample solution to be measured is introduced into a quantitative injection valve, a fixed amount of the sample solution to be measured is stored in the valve, and the flow path of the quantitative injection valve is switched.
A method of separately injecting a fixed amount of a sample into a liquid delivery system and detecting a specific compound after some separation and reaction is often used. Typical examples are liquid chromatographs and flow injection devices. However, in the case of a highly contaminated sample such as a fermented liquid, there has been a problem that the metering valve is clogged and the detector is contaminated and the accuracy is lowered. In order to prevent this, a device of the type that analyzes the filtrate by filtering the sample liquid to be measured with a filter so that microorganisms and solids contained in the sample liquid to be measured such as the fermentation liquid do not reach the detection unit has been often used. Has been. In this method, the filter is clogged, frequent replacement of the filter is difficult and maintenance is difficult, and there is also a method of disposing the filter as a single use, but it has a drawback of increasing the analysis cost. For this reason, separate liquids are allowed to flow on both sides of the membrane, and when the sample liquid to be measured and the liquid flowing on the other side of the film are in contact with each other across the membrane, they are dissolved in a part of the sample liquid to be measured or the sample liquid to be measured. There is an apparatus of a type in which a part of a chemical substance is transferred through a membrane and the transferred sample liquid or chemical substance to be measured is detected.

[0003]

The above-mentioned phase transfer method is less likely to cause clogging than the filtration method using a filter, but there is a possibility that a change in the measured value may occur due to a change in the characteristics of the membrane.
Conventionally, it has been difficult to measure with high precision. Further, it takes a long time for the solution or the substance to move through the membrane, which requires a long time for the measurement, and at the same time, the sample consumption amount increases. In addition, the amount of substance that can be redissolved is almost determined by the characteristics of the membrane, and the concentration range to be measured is determined not only by the dynamic range of the detector but also by the characteristics of the membrane, limiting the application range of the sample, or The concentration range that can be measured was limited, and the application range was narrow.

[0003]

According to the present invention, in the above-mentioned transfer type concentration measuring apparatus, an opening / closing mechanism for opening / closing a flow path at an arbitrary time is provided in the middle of a pipe for guiding a sample liquid to be measured to the separation mechanism. Open the sample liquid channel to be measured at an arbitrary fixed interval where the volume of the sample liquid to be measured fed to the membrane volume of the separation mechanism is 5 times the volume of the channel or more. The membrane and the sample solution to be measured were brought into contact with each other. By opening the sample liquid channel to be measured at regular time intervals such that the amount of the sample liquid to be measured to be fed is 5 times or more the flow channel volume,
In the separation mechanism, the relative velocity of the sample liquid to be measured and the liquid of a different type is constant, and there is a moment when the inside of the flow path is completely replaced by the sample liquid to be measured. By detecting the amount of inversion during this transient process, the sample amount can be minimized and accurate measurement can be performed. Further, by setting the time for contacting the sample liquid to be measured with the membrane so that the amount of liquid transfer is 5 times or more of the volume of the channel, the shorter the time is within the range of the conditions, the more the solution is transferred. The amount or amount of chemicals decreases and the sensitivity decreases. On the contrary, when the length is increased, the sensitivity is increased, and the sensitivity can be adjusted on the film surface. Further, the present invention comprises a pipe that alternately guides the standard solution and the sample liquid to be measured to the separation mechanism, and provides an opening / closing mechanism capable of opening and closing the flow path at an arbitrary point in the middle of each pipe, and the membrane surface of the separation mechanism. Open the standard solution and measured sample solution flow paths at an arbitrary fixed interval where the volume of the standard solution or measured sample solution sent to the flow path volume of the part that contacts Then, the sample solution to be measured was first contacted for a certain period of time, and then the detected value when the standard solution was contacted for a predetermined period of time was memorized, and then the sample solution to be measured was contacted with the standard solution for the same period of time. It was used to calculate the concentration by comparing with the detected value at the time. In this way, by contacting the sample solution with the film surface in advance, the standard solution and the sample solution under test are sequentially contacted under certain conditions after causing the film surface to fluctuate due to contact with the sample. As a result, it becomes possible to measure with higher accuracy.

[0005]

DETAILED DESCRIPTION OF THE INVENTION An embodiment of the present invention will be described with reference to FIG. 9 is the above-mentioned separation mechanism and 10 is a separation membrane. Reference numeral 6 is a pipe for the sample liquid sending system, which is the membrane 1 of the separation mechanism.
0 is connected to the upper flow path, an on-off valve is inserted in the middle, and a liquid feed pump 1 is provided on the upstream side. This pump may be provided on the downstream side of the on-off valve 4 as shown in FIG. First, the invention of claim 1 will be described assuming that the flow path 7 is not provided in FIG. Reference numeral 8 denotes a liquid sending pipe connected to the lower channel of the membrane 10 of the separation mechanism 9 and a liquid different from the sample liquid to be measured is detected by the pump 3 through the lower channel of the membrane 10 of the separation mechanism. It is designed to be sent to the container 11. The present invention uses such a device to measure the concentration as follows. That is, while the pump 3 is driven to flow a liquid such as a buffer solution into the lower flow path of the separation mechanism 9, the open / close valve 4 is used to cause the sample liquid to be measured to flow into the upper flow path of the separation mechanism at 5 times its volume or more. The output of the detector 11 is read during the period in which the output of the detector 11 is opened for a certain amount of time. The method of reading the output is, for example, to start data collection with the start point of contact of the sample with the film as the start point, and store the data up to the point where the output decreases after the contact with the film is completed. , Find the maximum and minimum points of output. Note that the output is averaged in the vicinity of the maximum and the minimum, and the S / N
Can also be improved. As shown in FIG. 1, the invention of claim 2 is a device in which a flow path pipe 7 is added to the above-mentioned device, whereby the sample solution to be measured and the standard solution are alternately flowed through the pipes 6 and 7 above the separation mechanism 9. First, the sample solution to be measured is allowed to flow through the separation mechanism for a certain period of time, and then the standard solution is allowed to flow through the upper path of the separation mechanism for another certain period of time, and the output of the detector at that time is stored. The sample liquid to be measured is allowed to flow for the same time as the standard solution, and the output of the detector at that time is compared with the output of the previously stored detector.

As a result of studying a method for making the amount of permeation of the compound to be measured in the separation mechanism part as constant as possible and improving the accuracy of the measurement result, the accuracy and sensitivity are in contact with the membrane regardless of the flow velocity and the shape of the flow channel. It was found that it is related to the volume of the flow channel and the amount of the sample liquid to be measured that has passed through the inside of the channel, and further studies have revealed that it is sufficient to pass the sample liquid to be measured at 5 times or more the volume of the channel. For example, in the case where the sample to be measured is glucose, if the membrane and the sample are in contact with each other in an area of 40 mm ² and the channel cross section is a square of 1 mm, the channel volume is 0.04 cm ³ , that is, 40 μl. In this case, at least 200 μl of the sample liquid to be measured may be passed. If the flow rate of the tube pump for feeding the sample liquid to be measured is 2 ml / min, accurate results can be obtained by feeding the liquid for 10 seconds. If, for example, a buffer solution is flowing at 2 ml / min on the opposite surface of the membrane, the concentration distribution of the transferred compound to be measured has a shape like the peak of a liquid chromatograph.
The fluctuation rate of the measured value at this time is 3%. However, when the volume of the sample liquid to be measured is three times the volume of the flow channel, the fluctuation rate of the measured value is as high as 10%. For example, the case where the sample solution to be measured is a yeast culture solution, the compound to be measured is glucose, and the detection unit is an enzyme electrode having glucose oxidase immobilized thereon will be described. The glucose solution was used as the sample solution to be measured, the membrane area was 40 mm ² , and the channel volume was 40
When 200 μl to 1000 μl of the sample liquid to be measured is brought into contact with the separation mechanism part of μl, the obtained detection value is proportional to the amount of the sample liquid to be contacted and increases almost linearly. That is, in the case of measuring low concentration with high accuracy, the volume of the sample liquid to be measured may be increased, and the sensitivity can be controlled by controlling the passage time of the sample liquid to be measured. Further, in the case of a fermentation broth, cells, various compounds derived from cells, impurities of the medium, etc. are contained, and the membrane permeability may change due to contact with the membrane surface for a long time, but the method of the present invention Since the contact time of the sample liquid to be measured is limited, this fluctuation amount is relatively small, but it is not completely ineffective. Therefore, first the sample liquid to be measured is brought into contact with the film surface for a short time to change the film surface state in the sample, and immediately after that, the standard solution is measured, and the detected value and the measured sample liquid after that are measured. The most accurate measurement can be made by comparing the detected values. Of course, there is no problem if a washing step with distilled water or the like is inserted between the sample solution to be measured and the standard solution, but in this case as well, it is necessary to carry out the washing step at regular intervals. In addition, in the step of contacting the sample liquid to be measured in advance, it is possible to discard the old sample liquid to be measured accumulated in the pipe and the like, and there is an advantage that unnecessary operation of the apparatus can be eliminated.

In the present invention, the material and thickness of the tubes constituting the piping systems 6, 7, 8 etc. are not particularly limited, but at least when the pinch valves described below are used as the opening / closing means 4, 5 etc. The portion sandwiched by the pinch valves is preferably an elastic body having a restoring force, such as a silicone tube, a vinyl chloride resin tube, or a polyolefin tube. Above all, a polyolefin tube is preferable because it has low gas permeability and air does not enter from the outer wall of the tube when the liquid is stopped and bubbles are not generated. When integrating these tubes, two-way to four-way joints are used. The structure of this portion is not particularly limited, but a method of attaching the tube to a joint made of a material harder than the tube such as metal or plastic is simple and preferable.
When the joint material is made of fluororesin, polypropylene, or polyethylene, it is easy to process and various shapes can be prepared, and corrosion due to a liquid flowing through like metal does not easily occur, which is preferable. Furthermore, the fluororesin and polypropylene joints are more preferable because they can be subjected to pressure steam sterilization when feeding the fermentation liquid. As the valve, various ones such as a pinch valve, an electromagnetic valve, a motor-driven multi-way valve, etc. are used, but it is necessary to select the internal pipe diameter in order to prevent clogging due to turbidity of the sample liquid to be measured. The pinch valve is most advantageous in the point that the piping used is used for closing the flow path as it is.

The description of the basic operation and piping system used in the concentration measuring method of the present invention will be supplemented. One-sided flow of a membrane of a separation mechanism equipped with a membrane, where the piping systems 6 and 7 equipped with a pinch valve are joined using a joining joint 12 installed on the downstream side with respect to the liquid flow direction of the pinch valve installation part. Guide the sample solution to be measured into the channel. The pumps 1 and 2 may be placed before the joint 12 as shown in FIG. 1, after the joint 12 as shown in FIG. 2, or after the separating mechanism 9.
In general, when the flow path inside the separation mechanism is narrow and the back pressure at the time of passage may increase, it is better to put it in front of the separation mechanism to introduce pressure. Various pumps such as gear pumps, plunger pumps, and tube pumps can be used here, but for the purpose of sending a constant measurement sample solution containing turbidity at low cost, the tube is squeezed to send the solution. Performing tube pumps are preferred. As the tube used for the tube pump, various kinds of tubes can be used in the same manner as the tube used for the above-mentioned piping, but a polyolefin-based tube capable of obtaining long-term liquid transfer rate stability is preferably used.

Examples of the membrane generally used in the separation mechanism having a membrane include ultrafiltration membranes, dialysis membranes and membrane filters. As the ultrafiltration membrane, various membrane materials can be used depending on the substance to be separated and measured, and examples thereof include polysulfone membrane and cellulose acetate membrane. A regenerated cellulose membrane or the like is used as the dialysis membrane. As the membrane filter, those made of fluororesin, polycarbonate, regenerated cellulose, nitrocellulose, etc. are used. When measuring water-soluble small molecules,
A durable dialysis membrane that does not require pressurization is preferred.
In addition, when vaporizing and transferring the dissolved ammonia and alcohol in water, if you use a fluororesin membrane filter with a filter pore size of 0.1 to 1.0 μm, it becomes a kind of gas permeable membrane and only the target substance is transferred. More effectively preventing unwanted components from reaching the detector. The separation mechanism itself can be made of any material such as metal or plastic, but in order to prevent corrosion by liquid, stainless steel,
It is preferably made of titanium material, acrylic resin, fluororesin, polypropylene, polyethylene, polycarbonate or the like. In particular, it is desirable to manufacture it with plastic because it does not damage the membrane when the dialysis membrane is sandwiched.

The detector used to detect the substance to be measured is an absorptiometer, a fluorometer, a pH meter, an ion electrode, a semiconductor ion sensor, an electrochemical detector, an atomic absorption analyzer, an induction plasma emission analyzer, an enzyme. Known detectors such as electrodes and thermometers are used. It is also possible to cause a chemical reaction inside the flow channel and detect the physical quantity that changes as a result, and after removing the bacterial cells of the fermentation liquid, introduce it into a separation column and connect it to a so-called liquid chromatograph. It is also possible. Of these, the use of an enzyme electrode capable of specifically detecting a specific component is desirable in that the device can be configured easily and the accuracy can be easily increased. As for the type of enzyme electrode, hydrogen peroxide electrode, oxygen electrode, pH electrode, and other types in which the enzyme is immobilized on the surface of the membrane, but the enzyme immobilized on the carrier is packed in the column and various electrodes are placed downstream of it. May be installed. As an enzyme immobilization method, various known methods such as a chemical binding method, an entrapping method, and an ionic binding method can be used. Further, when immobilizing the enzyme in the form of a film on the electrode, the electrode surface may be in direct contact with the enzyme or a compound that crosslinks with the enzyme or enzymes such as albumin and enzyme,
The enzyme may be immobilized on the acetyl cellulose membrane and held by pressing the membrane against the electrode surface. When immobilized on a carrier, known carriers such as activated carbon, silica gel and diatomaceous earth can be used. The method of using a carrier as a column is advantageous because a large amount of protein can be immobilized when the enzyme activity is low.

[0011]

EXAMPLES Examples of the present invention are shown below, but the present invention is not limited thereto.

Example 1 As shown in FIG. 1, the outer diameter is 4 mm and the inner diameter is 2
mm silicon tube, pinch valves 4 and 5, and a first measured sample solution sending system 6 including a first measured sample solution sending pump 1 and a second tested sample solution sending system 6 having the same structure. The measurement sample liquid system 7 was connected by a polypropylene 3-way joint. The sample liquid to be measured is a tube pump for liquid transfer 1
Both and 2 are suction-fed at a flow rate of 2 ml / min. Either the pinch valve 4 or 5 is opened to switch the sample liquid to be measured. When the pinch valve was opened, the tube pump arranged in the piping system was driven to switch the sample liquid to be measured. The separation mechanism 9 is equipped with a regenerated cellulose dialysis membrane 10 having a film thickness of 20 μm, and on the opposite side of the membrane, a third liquid delivery tube pump 3 is used to adjust the pH to 7.0.
1M phosphate buffer is running. The volume of the flow channel facing the membrane of the separation mechanism 9 was 120 μl. Detector 11 equipped with an enzyme electrode on which glucose oxidase is immobilized
Was provided downstream of the separation mechanism 9. As the sample liquid to be measured,
Fermentation model liquids containing 1% yeast cells each having a 1.8 (WT / V)% glucose concentration and a 3.6 (WT / V)% glucose concentration were used. 30 when the pinch valve is opened
Seconds, the amount of each sample liquid to be measured to be sent was about 8.3 times the flow channel volume, and 1000 μl was sent. This sample was alternately fed 5 times each, and the glucose concentration of the fermentation model liquid was calculated from the value at the detector 11 at that time. Table 1 shows the results.

[Table 1]

[0012]

Example 2 As shown in FIG. 2, the outer diameter is 4 mm and the inner diameter is 2 mm.
mm silicon tube, pinch valves 4 and 5, and a first measured sample solution sending system 6 including a first measured sample solution sending pump 1 and a second tested sample solution sending system 6 having the same structure. The measurement sample liquid system 7 was connected by a polypropylene 3-way joint. The sample liquid to be measured is a tube pump for liquid transfer 1
Is aspirated at a flow rate of 2 ml / min. Either the pinch valve 4 or 5 is opened to switch the sample liquid to be measured. The experiment was conducted by setting the time for opening the pinch valve to 45 seconds. Separation mechanism 9
Is equipped with a regenerated cellulose dialysis membrane 10 having a thickness of 20 μm, and a 0.1 M phosphate buffer solution having a pH of 7.0 is caused to flow by a third tube pump 3 for delivering liquid on the opposite side of the membrane. ing. The flow path volume facing the membrane of the separation mechanism 9 is 200
μl. A detector 11 equipped with an enzyme electrode on which glucose oxidase was immobilized was provided downstream of the separation mechanism 9. As the sample liquid to be measured, a fermentation model liquid having a glucose concentration of 0.9 (WT / V)% and containing 1% of yeast cells was used, and as a standard liquid, an aqueous solution of 1.8 (WT / V)% glucose was used. Using. After sending the sample liquid to be measured in advance for 20 seconds,
The standard solution and the sample solution to be measured are sequentially sent to the separation mechanism 9 for 45 seconds respectively, and the sample solution calibrated is calibrated based on the value of the standard solution obtained by the detector 11 at that time and the value of the sample solution to be measured thereafter. Calculate the glucose concentration of. This step was repeated five times. The standard solution and the sample solution to be measured were sent in an amount of 1500 μl, which is about 7.5 times the flow path volume. This sample was alternately fed 5 times, and the glucose concentration of the standard solution fermentation model liquid was calculated from the value at the detector 11 at that time.

[0013]

Comparative Example 1 The same experiment as in Example 2 was carried out except that the sample solution to be measured was sent to the separation mechanism 9 for 20 seconds before sending the standard solution. These results are shown in Table 2 together with the results of Example 2. In Example 2, since the standard solution and the sample solution to be measured are successively contacted with each other under a certain condition after the film surface was changed in advance due to the contact with the sample solution to be measured, the quantification accuracy was better and the reproducibility was also good. It turned out to be good.

[Table 2]

[0014]

By using the concentration measuring device of the present invention, it becomes possible to measure a sample such as a fermentation liquor which is easily contaminated with a simple structure with a small volume and with high accuracy.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a concentration measuring device used in Example 1 of the present invention.

FIG. 2 is a schematic view of a concentration measuring device used in Example 2 of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... 1st pump for sending sample liquid to be measured 2 ... 2nd pump for sending sample liquid to be measured 3 ... Pump for sending buffer solution 4 ... 1st pinch valve 5・ ・ ・ Second pinch valve 6 ・ ・ ・ First sample liquid supply pipe 7 ・ ・ ・ Second sample liquid supply pipe 8 ・ ・ ・ Buffer liquid liquid supply pipe 9 ・ ・ ・ Separation mechanism 10 ・..Separation membrane 11 ... Detector having enzyme electrode

Claims (2)

[Claims] 1. A liquid-feeding mechanism that has a membrane and has a separation mechanism that allows separate liquids to flow on both surfaces of the membrane, and a liquid-feeding mechanism that brings a sample liquid to be measured into contact with one surface of the membrane of the separation mechanism, and a separate mechanism of the membrane. It has a separate liquid transfer mechanism that allows the liquid flowing in contact with the surface to flow, and when the sample liquid to be measured and the liquid flowing to the other surface of the film are in contact with each other across the membrane, part of the sample liquid to be measured or the sample to be measured A part of the chemical substance dissolved in the liquid is transferred through the membrane, and a concentration measuring device equipped with a detector that detects the transferred sample liquid or the chemical substance is used to separate the sample liquid to be measured. An opening / closing mechanism that can open and close the flow path at any time is provided in the middle of the pipe leading to the flow path, and the volume of the sample liquid to be measured that has been sent to the flow path volume of the portion that contacts the membrane surface of the separation mechanism is the flow path. Open the sample liquid flow path to be measured at an arbitrary fixed time interval that is 5 times or more of the volume, and connect the membrane and the sample liquid to be measured. A method for measuring concentration, which comprises contacting and calculating the concentration from the output of the detection unit during this period. 2. A pipe for guiding the standard solution and the sample liquid to be measured to a separation mechanism is provided, and this pipe is integrated into one pipe before entering the separation mechanism having a membrane, and flows in the middle of each pipe. By using a device equipped with an opening / closing mechanism that can open and close the channel at any time, the volume of the standard solution or the sample solution to be measured is flowed to the volume of the channel of the part in contact with the membrane surface of the separation mechanism. Open the flow path of the standard solution and the sample solution to be measured at an arbitrary fixed interval that is more than 5 times the channel volume, first contact the sample solution to be measured for a certain period of time, and then contact the standard solution for a certain period of time specified separately. 2. The concentration measuring method according to claim 1, wherein the detected value at the time of storage is stored, and then the concentration is calculated by comparing with the detected value when the sample solution to be measured is brought into contact with the standard solution for the same time.