JP2003270007A - Method and device for measuring minute flow rate and method of reproducing measuring tube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pump in which a minute flow rate not more than flow rates of mL/min to μL/min can be accurately and easily measure, and of while measure is to easily measure a minute flow rate pump, and to provide pump capable of accurately pumping out the minute flow. <P>SOLUTION: The minute flow rate pump 1 that is an object to be measured is set at the edge of a path B, and the path B connects a resistance tube 2 and a sample injector 3 to a sampling tube 6, and a microscope 4 is set so as to be opposite to the sampling tube 6. Then, carrier containing a fluorescent reagent is pumped out from the minute flow rate pump 1, and bubbles are injected from the sample injector 3. The carrier corresponding to a space between a mark point of the sampling tube 6 and an arrival point where the bubbles move for a prescribed period, is sampled, and the intensity of fluorescence is quantified, thereby measuring a reduced flow rate. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring method and device for a minute flow rate and a measuring tube duplication method.

[0002]

2. Description of the Related Art Conventionally, a flow rate measurement has been stipulated as a method in the measuring method to measure the weight by a balance primarily, and is substantially performed by using a microsyringe or a graduated cylinder that is fair in the balance method. ing. Above all mL / m
For the flow rate of in to several μL / min, a graduated cylinder, a ball pipette, a microsyringe or the like was used, and the flow rate was measured using the scale.

Further, along with the miniaturization of HPLC, a pump for accurately feeding a minute flow rate is required, and a technique for measuring the flow rate is required. The flow rate measurement in the low flow region (μL) of this minute flow rate pump was usually limited to a measurement up to 5 μL / min using a microsyringe.

However, there is an example described in Japanese Patent Laid-Open No. 10-114394 as a device for distributing a small amount of transferred fluid. This is to fill a microdispenser and a microdispenser using a piezoelectric transducer attached to a glass capillary tube with a transfer fluid, suck the transfer fluid from the microdispenser, control the pressure of the system fluid, and transfer and transfer the fluid. Measuring the pressure of the system fluid with a positive displacement pump to wash the microdispenser between
And a pressure sensor that emits a corresponding electrical signal. In short, a micro dispenser dispenses a small amount of fluid by a pressure signal.

As the splitter method, a method of supplying an extremely small amount of flow is known, although it is not a method of measuring the flow. This is to accurately split the solvent sent from the primary pump and to supply it, and to supply a stable solvent etc. to the system such as micro LC, LC / MS at a micro flow rate by using a semi-micro compatible pump etc. together. Suitable to do.

Further, a fluorescence method is used as a means for detecting and measuring a trace amount of concentration. The fluorescence detector measures the fluorescence energy of a substance excited by irradiating ultraviolet rays. Since it has a high detection sensitivity and can be measured by a relatively inexpensive equipment, for example, a cell measurement required portion (gene DN
(Including A etc.) is marked with a fluorescent substance, a certain reaction is performed, the amount and position of the fluorescent substance after the reaction is measured, and further, it is generally used as a detector for liquid chromatography. .

[0007]

The measurement of the flow rate using a graduated cylinder, a microsyringe, etc. using the scale was difficult and inaccurate at a flow rate of several μL / min or less. However, as the flow velocity becomes slower, it is difficult to accurately visually recognize the behavior of the liquid level passing through the scale of the syringe, and it is doubtful whether the absolute value of the flow rate can be obtained strictly due to individual differences in visual recognition.

Further, when a minute flow rate pump is used, the only reliable method for measuring the flow rate is the measurement up to 5 μL / min. Therefore, the pump flow rate for accurately feeding the minute flow rate cannot be verified. There is a demand for the flow measurement technology. On the other hand, it is pointed out that importance is attached to a method and a device for supplying an extremely minute flow rate, which is valuable in the field of biotechnology, a micro flow pump as a liquid feeding means, and a measurement of a liquid flow rate of the micro flow pump.

The device described in Japanese Patent Laid-Open No. 10-114394 is excellent in that the transferred fluid is measured and distributed, but it is a large-scale device for measuring the flow rate of the fluid, and its manufacturing cost is extremely high. . Therefore, it is difficult to use it easily anytime and anywhere. Also, in the splitter method, the set flow rate is not obtained mechanically,
Since the flow rate largely depends on the content resistance of the resistance tube, it is experimentally known that the obtained flow rate is easily affected by temperature. Therefore, it is necessary to perform the measurement under the environment where the room temperature is constant. Furthermore, if the composition of the solvent changes (mandatory operation in the gradient analysis of liquid chromatography) or the temperature changes, the volume changes. Therefore, it is unavoidable that the flow rate fluctuates by about 3%. The fluorescence method is used to measure the concentration and molecular weight of substances,
It is used to detect metabolites, vitamins, amino acids, amines, steroids, etc.

[0010]

Therefore, in the present invention, it is easy to measure a flow rate of several μL / min or less,
In addition, we propose a method that can accurately and accurately measure the pump flow rate in the minute flow rate range of mL / min to several μL / min. First, the fluorescent reagent is placed in the sampling tube for a certain period of time. It is characterized in that the converted flow rate is calculated by sending the carrier containing the solution and quantifying the amount of the solution carried out by fluorescence, and secondly, during the sending of the carrier containing the fluorescent reagent into the sampling tube, bubbles are generated from a certain point. The feature is that the converted flow rate is calculated by quantitatively measuring the fluorescence intensity of the carrier amount between the mark point on the sampling tube where the bubbles flow and the arrival point after a predetermined time has passed. A target object such as a pump to be installed can be freely installed, a resistance tube and a sample injector are connected by a sampling tube, and a microscope is installed at a desired position of the sampling tube. In addition, a sampling tube can be freely installed in the path terminal. Fourthly, a micro flow pump is installed at the end of the path, and a resistance tube, a sample injector, and a microscope are installed in the path, and the path terminal is provided. While the sampling tube can be freely installed in the sample tube, bubbles are injected while the carrier is being sent from the pump at a constant flow rate, and the sampling tube having a constant internal volume is obtained by marking the bubble position after a lapse of a certain time from the injection mark position. Fifthly, a minute flow rate pump is installed at the end of the path, a first sample injector and a microscope are installed, and a second sample injector and a microscope are installed at appropriate intervals in the path. Install the measurement sampling tube under the field of view of the first sample injector and microscope. Air is sent from the two sample injectors, and the bubbles injected into the measurement sampling tube pass through the mark of the start position and at the same time mark the position of the bubbles in the sampling tube, and the bubbles of the measurement sampling tube end position. The feature is that the bubble position of the sampling tube is marked at the same time when the mark passes.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, according to the embodiment shown in the drawings,
The present invention will be described in detail. Reference numeral 1 is a minute flow rate pump to be measured, which is used as a carrier for liquid delivery depending on the type of detector. When a fluorescence detector is used, it is convenient to detect the fluorescence intensity of quinine sulfate. Reference numeral 2 is a resistance tube for stabilizing the carrier, and a capillary tube is used to eliminate dead volume. For example, a fused silica capillary tube, a glass capillary tube, a stainless capillary tube or the like is used. Numeral 3 is a sample injector, and it is convenient to use a sample injector having a desired number of needle ports, and a sample rotor having an appropriate capacity from 0.1 μL is selected and used. Reference numeral 4 denotes a microscove, and a marking set 5, for example, a device for marking down the sampling tube 6 by a switch or manual lowering of Magic Ink (registered trademark) is provided under the field of view, or a monitor of the microscope 4 is provided. Mark by hand while looking. It is convenient to use the same capillary tube as the resistance tube 2 as the sampling tube 6. Various capillary tubes may be used as the resistance tube 2 and the sampling tube 6, but the fused silica tube is convenient because it is easy to handle and cut. Resistance tube 2
Has a diameter of 0.010 to 0.050 mm and a sampling tube 6
0.020 to 0.100 mm is convenient for use.

Next, the measuring method will be described. First, the minute flow rate pump 1 to be used for measuring the flow rate is connected to the measuring device A including the resistance tube 2, the sample injector 3, the microscope 4, and the sampling tube 6.
Next, the carrier is sent from the minute flow rate pump 1, and the resistance tube 2 and the sample injector 3 are connected by the sampling tube 6. The route B is filled. A 0.1 M aqueous sulfuric acid solution containing quinine sulfate is used as the carrier. A digital microscope is convenient as the microscope 4.

Next, while observing the monitor (not shown) of the microscope 4, the magic ink (registered trademark) is applied to the surface of the sampling tube 6 under the visual field of the microscope 4.
Mark with etc. In this operation, when the marking set 5 is used, the marking is performed mechanically, or in the case of manual operation, the marking is performed manually. The liquid feeding of the carrier is started in accordance with the set flow rate of the minute flow rate pump 1 and left until the back pressure becomes stable. When the back pressure becomes stable, air (air bubbles) is injected from the needle port of the sampling injector 3 using a syringe to fill the inside of the sample rotor with air. The sampler used was 0.2 μL.

After that, the valve of the sample injector 3 is switched to the INJECT mode, and the bubble X is injected into the sampling tube 6. The microscope 4 observes how the bubble X injected into the sampling tube 6 passes through the mark 61 previously marked with Magic Ink (registered trademark), and measures the elapsed time from the time when the bubble X passes through the mark 61. Start. After a lapse of a predetermined time, the sampling tube 6 is cut with a magic mark 61, and its cut 62 is quickly put into the measuring flask 7 (volume 10 ml). Next, the air bubble X portion of the sampling pipe 6 is cut, and the cut end 63 is connected to the pump 10 through the connecting pipe 9 such as a PEEK tube using the union 8. At this time, the volumetric flask 7 is preferably covered with the film 11 or the like.

Subsequently, the carrier (quinine sulfate aqueous solution) in the sampling tube 6 is discharged into the measuring flask 7 by sending an excessive amount of 0.1 M sulfuric acid from the pump 10 to clean the inside of the sampling tube 6. The discharged quinine sulfate is made up to 10 ml with 0.1 M sulfuric acid. The fluorescence intensity of the quinine sulfate is quantified using a fluorescence detector, and the volume in the cut measurement sampling tube 60 is accurately obtained to convert the flow rate. From the calibration curve regression equation obtained from the quinine sulfate standard solution, the content is converted and further converted into the flow rate. (Table 6)

The experimental results measured under the measurement conditions shown in Table 1 are shown below.

[Table 1] [Experiment 1] Measurement result at a set flow rate of 5 μL / min

[Table 2] [Experiment 2] Measurement result at a set flow rate of 1 μL / min

[Table 3] [Experiment 3] Measurement result at a set flow rate of 0.5 μL / min

[Table 4] [Experiment 4] Measurement result at a set flow rate of 0.1 μL / min

[Table 5]

[Table 6] The results obtained above were good reproducibility for each flow rate, and were very small from the set flow rate value of the pump. From these results, it is presumed that the liquid flow rate of the pump to be measured is accurate and that the result of this measurement method is also accurate. In addition, this measurement method is basically based on the fluorometric method, and since no complicated operations other than the fluorometric operation are performed, its accuracy is equivalent to that of the fluorometric method. It is speculated that there is.

Next, a method of duplicating a measuring tube, that is, a measuring sampling tube used in the above measuring method and apparatus will be described. First, the first is a method using the measuring apparatus A of FIG. As the pump 1, a minute flow rate pump whose flow rate is verified by the above-mentioned measurement method is used.
A carrier, for example, an aqueous solution of quinine sulfate is fed from the above, and after waiting for the back pressure to stabilize, the bubble X is injected from the sample injector 3, and the passage point of the bubble X is marked by the microscope 4 or monitor and passed. From time to time, the arrival point of the bubble X after a desired fixed time has elapsed is marked, and the sampling tube 6 such as a fused silica capillary tube is cut at both marking points to obtain a capillary tube with a constant internal volume, that is, a measurement sampling tube. Can be produced.

Next, the second method will be described with reference to FIG. The same tumbling injector 12, microscope 13, and marking set 14 as in FIG. 1 are installed downstream of the sampling tube 6 in FIG. Under the visual field of the microscope 4, a calibrated measurement sampling tube 65 with marks of start and end positions is installed at both ends, and is connected to the sampling tube 6 such as a fused silica capillary tube. Water or an appropriate liquid (methanol or the like) is fed as a carrier from the pump 1 to fill the entire route B. Start the liquid transfer according to the set flow rate of the pump 1, and keep it until the back pressure becomes stable.

Air is injected from the needle ports of the two sample injectors 3 and 12 using a syringe,
Fill the sample rotor with air. Then, the valve of the sample injector is switched to the INJECT mode, and the microscope 1 of the sampling tube 6 such as the standard capillary tube 65 and the fused silica capillary tube 1 is switched.
3 Air (air bubbles) is injected under the visual field. At the same time that the air bubbles that have entered the standard capillary tube 65 pass the start position mark, a mark is attached to the position of the sampling tube 6 under the visual field of the microscope 13.

Next, the bubble injected into the standard capillary tube 65 passes through the mark at the end position, and at the same time, the bubble position of the sampling tube 6 under the field of view of the microscope 13 is marked. As a result, two marks are attached to the sampling tube 6, and the measured sampling tube 65 having the same content as the measuring sampling tube 65 is duplicated by cutting both points.

[0021]

EFFECTS OF THE INVENTION According to the first to fourth aspects of the present invention as described above, it is possible to extremely accurately and easily measure the liquid sending flow rate in the minute flow rate range of mL / min to several μL / min. Moreover, the apparatus for the measurement is extremely simple, easy to manufacture, and easy to handle, which is a measure that contributes to the measurement of minute flow rates. Furthermore, pumping can be performed accurately and reproducibly in this minute flow region. Therefore, it is possible to provide a minute flow rate pump having an accurate flow rate. This method does not use a fixed-volume instrument with a scale as a sampling container as in the past, but the internal volume can be freely changed by adjusting the inner diameter and length of the capillary tube, so various methods are possible. It can be applied to flow rate measurement. Furthermore, if necessary, 0.1 μL / m
It seems that it is possible to measure the flow rate of in or less.

Further, according to the fifth and sixth aspects of the present invention, the minute flow rate measuring tube can be manufactured and provided extremely simply and easily, and the gap between the capillaries similar to that of the standard tube whose flow rate is verified can be easily and easily provided. It can be manufactured quickly.

[Brief description of drawings]

FIG. 1 is a schematic explanatory view of an apparatus according to an embodiment of the present invention.

FIG. 2 is an enlarged explanatory view of a main part of the present invention.

FIG. 3 is an enlarged explanatory view of a main part of the present invention.

FIG. 4 is a schematic explanatory view of an apparatus according to another embodiment of the present invention.

FIG. 5 is an explanatory view of the flow path on the sample injector side.

FIG. 6 is an operation explanatory diagram of the same as above.

[Explanation of symbols]

1 pump 2 resistance tube 3 sample injectors 4 Microscope 5 marking set 6 sampling tubes

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toru Kobayashi             Fukushima Prefecture Fukushima City Okajima character 5-3 Chocho             Le Science Co., Ltd. Fukushima Factory F-term (reference) 2F030 CA02 CC01 CF07

Claims (6)

[Claims] 1. A method for measuring a minute flow rate, which comprises calculating a converted flow rate by sending a carrier containing a fluorescent reagent into a sampling tube for a certain period of time, and quantifying the amount of the solution sent by fluorescence. 2. A carrier amount between a mark point on the sampling tube where the bubble flows and an arrival point after a predetermined time elapses, when bubbles are injected from a fixed point while the carrier containing the fluorescent reagent is being fed into the sampling tube. A method for measuring a minute flow rate, characterized in that the converted flow rate is calculated by quantifying the fluorescence intensity. 3. The carrier between the mark point on the sampling tube and the arrival point after a lapse of a predetermined time is stored in a container and quantified by cutting the sample tube between both points. Alternatively, the method for measuring a minute flow rate according to claim 2. 4. An object such as a pump to be measured can be freely installed in the path, and the path connects a resistance tube and a sample injector with a sampling tube, and a microscope is installed at a desired position of the sampling tube. A micro flow rate measuring device characterized in that a sampling tube can be freely installed in the terminal. 5. A micro-flow pump is installed at the end of the path, a resistance tube, a sample injector, and a microscope are installed in the path, and a sampling tube is freely installed at the end of the path. A method of replicating a measurement sampling tube, wherein a bubble is injected while feeding a carrier, and a sampling tube having a constant internal volume is obtained by marking the bubble position after a lapse of a fixed time from the mark position at the time of injection. 6. A micro flow pump is installed at the end of the path, a first sample injector and a microscope are installed, and a second sample injector and a microscope are installed at appropriate intervals in the path. 1 sample injector, a measurement sampling tube is installed under the microscope field of view, air is sent from two sample injectors, and the bubbles injected into the measurement sampling tube pass through the mark at the start position and A method for replicating a measurement sampling tube having a constant inner volume, which comprises marking a position and further marking the bubble position of the sampling tube at the same time when the bubbles of the measurement sampling tube pass through the mark at the end position.