JPH0484771A - Flow type diluter - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a dilution device that is applicable to a flow-type measuring device, and relates to a flow-type dilution device that can perform quick and accurate measurements with a simple configuration. It is something.

(Prior Art) Conventionally, a fixed amount of a sample is injected into a continuous flow and guided to a spectrophotometric needle, an atomic absorption spectrometer equipped with a flow cell, an electrochemical detector, etc., to detect the analyte in the sample. Flow injection analysis methods for quantifying substances are known.

In the flow injection analysis method, operations such as mixing, separation, and chemical reactions, which have been performed manually by humans, can be performed in a continuous flow, thereby eliminating individual differences among the measurers as much as possible.

However, in order to ensure that the sample to be quantified is sufficiently within the quantification range of the detector, it is necessary to dilute the sample at a certain magnification in advance.

When the dilution operation is performed manually, it is necessary to take a fixed amount of each sample and diluent and mix them to form a solution with a uniform concentration. These operations require a considerable amount of time, and there are problems in that they are subject to errors due to the instruments and containers used, as well as human errors.

For this reason, automatic diluters have been proposed that use mechanical operations to simulate dilution operations performed by humans.

However, in this type of automatic diluter, the speed at which the sample and diluent are separated, mixed, and stirred is limited due to the handling of the liquid, and it takes a considerable amount of time. In particular, in order to uniformly mix the sample and the diluent, there has been a drawback that repeated stirring operations or sufficient stirring time are required. Moreover, it is extremely difficult to perform dilution at high speed because it is necessary to thoroughly clean the needle used during dilution and the inside of the pipe connected to the needle. Furthermore, the multiple collection/dispensing and washing steps are disadvantageous in terms of measurement accuracy.

In addition, the device itself has a complex structure and many parts, such as driving the needle up and down, left and right, front and back, driving the syringe to quantify the liquid, and also requiring a container to store the diluted solution in addition to the bottle to hold the sample. is necessary.

(Problems to be Solved by the Invention) The present invention provides a flow-through method that enables continuous, quick and accurate dilution with a simple device configuration without requiring mechanical separation and dispensing operations. The purpose of the present invention is to provide a type dilution device.

(Means for Solving the Problems) The present invention includes a first pump (1) for feeding a first carrier, a sample injection section (4), and the sample injection section (4).
) and a detection section (7), and includes a flow dilution device (5) downstream of the sample injection section (4) on the main flow path, and a flow dilution device (5) that connects the sample injection section (4) to the main flow path. An extraction channel and a second pump (
2), and further includes a confluence section (6) downstream of the branch section (5) on the main flow path, and a second confluence section (6) in the confluence section (6).
This is a flow type diluter characterized by having a confluence channel and a third pump (3) for feeding the carrier.

Further, in the present invention, it is preferable to provide a mixing pipe that generates turbulent flow within the pipe between the sample injection part (4) and the flow dividing part (5), and further, the mixing pipe is filled with granular material inside. It is preferable that the tube is made of aluminum.

(Operation) This will be explained below using the example shown in FIG. The first carrier is continuously fed to a sample injection part (4) of an autosampler or the like by a first pump (1). Sample injection part (4
), it is possible to inject the sample in the undiluted state.

The injected sample is carried by the first carrier to the downstream dividing section (5), where a fixed amount is continuously suctioned and separated from the main channel by the second pump. This separation substantially reduces the amount of sample and carrier carried.

In the present invention, the main flow path is from the sample injection section (4) to the detection section.
means the flow of liquid and its piping.

Furthermore, each pump actively generates a single liquid flow, and for example, to reduce pulsation, multiple pumps may be connected in parallel to generate a single liquid flow. A so-called multi-head type pump having the above structure is also included in the pump of the present invention.

Then, after a part of the first carrier has been suctioned and separated, the remainder proceeds through the main flow path and is sent to the downstream merging section (6).

At this junction (6), the second carrier is continuously added to the main channel at a constant rate by the third pump (3) to dilute the sample.

The second carrier added by the third pump (3) performs dilution and increases the flow rate of the main flow path, which has been reduced by being sucked and separated by the second pump (2), and is sent to the downstream detector (7). It also plays the role of feeding liquid and adjusting the passing speed. In other words, it is possible to adjust the observation time of the waveform observed using the flow injector analysis method and perform measurements at high speed.

In the flow type diluter of the present invention, the dilution rate can be adjusted by adjusting the liquid feeding speed of each pump. For example, to increase the dilution rate, the first pump (
The second pump (2
), or adjust the liquid delivery speed of the third pump (3) to be increased. However, in order for the sample to reach the detection part (7), the second
The extraction speed of the first pump (2) must be at least lower than the liquid delivery speed of the first pump (1).

It is also possible to reduce air bubbles mixed into the main flow path in the flow dividing section (5). In particular, if the second pump (2) is used to perform suction with the suction direction facing upward, air bubbles that have entered the main flow path can be efficiently removed and troubles caused by air bubbles generated in the detection section (7) can be prevented. There are advantages that can be achieved.

Examples of the detection unit (7) connected to the flow type diluter of the present invention include a spectrophotometer equipped with a flow cell, an atomic absorption spectrometer, or an electrochemical detector.

Examples of carriers used include organic solvents, buffer solutions, and reaction reagent solutions.

By using an electrochemical detection system that uses an immobilized enzyme such as an enzyme electrode in the detection section, it is possible to selectively analyze the target component in the sample.In this case, the first carrier and second carrier used A buffer solution can be used, and it can also be supplied from a common container.Also, it is also possible, for example, to use distilled water as the first carrier and a buffer solution as the second carrier, and this method This method is effective in reducing buffer consumption and lowering analysis costs.

Basically, the sample can be injected as an undiluted solution, but if solids or suspended matter are present, they may clog the pipes and cause significant fluctuations in the flow rate of the system. It is therefore advisable to filter it beforehand.

In order for the flow type diluter of the present invention to function in a more stable state, it is desirable that the liquid delivery rate of each pump be stable and that the pulsation be as low as possible. To this end, in order to apply appropriate pressure to each flow path and eliminate local pressure fluctuations, pressure adjustment piping can be provided before and after the detector, before and after the branching section, or before and after the merging section. The method for arranging the pressure regulating piping may include a method of using piping with the same diameter as the main flow path and increasing its length, a method of using piping with an inner diameter smaller than that of the main flow path, and the like. In addition, when the length is long, the piping can be wound into a coil to reduce the installation space.

Further, a pulsation damper may be provided after the liquid pump is discharged.

Although a manual injector or an autosampler can be used as the sample injection unit (4) connected in the present invention, it is preferable to use an autosampler in terms of automation and accuracy.

Furthermore, in systems such as alcohol and water, in which the sample and carrier are difficult to mix, as the concentration of alcohol increases, it tends to become difficult to mix with water. In measurements of such a system, it is desirable to provide a mechanism for promoting mixing of the sample and carrier between the sample injection section (4) and the flow dividing section (5).

Specifically, we installed a small-capacity mixing chamber with a stirrer,
Mixing piping can be provided that disrupts the flow of sample and carrier, creating turbulence within the tube. As the mixing piping, tubes made of stainless steel, fluororesin, vinyl chloride, etc. can be used. Among these, one mixing method that has a simple mechanism and provides high precision is the use of a tube filled with granules. This method allows the sample and carrier to be mixed effectively.

Further, a mixing pipe may be incorporated between the confluence section (6) and the detection section (7).

(Example) The present invention will be explained in more detail with reference to Examples below.
Of course, the present invention is not limited to these. In addition, what is simply written as % represents weight %.

Example 1 (1) Structure of Detection Section As the detection section (7), a glucose detector schematically shown in FIG. 2 was used. That is, an immobilized enzyme electrode (b) created by immobilizing glucose oxidase on a platinum electrode.
The details of the glucose detection unit are shown below, in which the flow cell incorporating the unit is arranged in a constant temperature bath OD and glucose is detected by electrochemical detection using a potentiostat 07).

a. The side surface of the immobilized enzyme electrode, a platinum wire with a diameter of 2 mm, was covered with heat-shrinkable Teflon.
Finish one end of the wire smooth with a file and 1500 emery paper. This platinum wire was used as a working electrode, a 1 cm square platinum plate was used as a counter electrode, and a saturated calomel electrode (hereinafter abbreviated as SCE) was used as a reference electrode. +2. in 1M sulfuric acid. Electrolytic treatment is performed for 5 minutes in OV. , then remove the platinum wire (after washing with water,
It was dried at 0° C. for 10 minutes, immersed in an anhydrous toluene solution of 10% T-aminopropyltriethoxysilane for 1 hour, and then washed. An enzyme was immobilized on this aminosilanized platinum wire as follows.

Glucose oxidase (manufactured by Sigma, type II) 5
mg, and bovine serum albumin (manufactured by Sigma, Frac
ion V) is dissolved in 1 ml of 100 mM sodium phosphate buffer (pH 7), and glutaraldehyde is added to a concentration of 0.2%. 5 μl of this mixed solution was quickly placed on the platinum wire prepared earlier, and dried and hardened at 40° C. for 15 minutes.

Then, 100mM sodium phosphate buffer (PH6)
Save inside.

b. Detection method: The immobilized enzyme electrode prepared in a above is used as a working electrode on the flow cell side, and the Ag/AgC1 reference electrode 09 is installed as a reference electrode in a direction perpendicular to the flow channel so as to face it. The stainless steel used in the above was attached as a counter electrode (161). A voltage of +0.6 V was applied to the immobilized enzyme electrode (2) by a potentiostat 0'7) with respect to the Ag/AgC1 reference electrode 05). These are 37°
It is installed inside a constant temperature bath OD maintained at ±0.2°C.

The detected current value is converted into current/voltage by the potentiostat 07), and this voltage value is taken into the computer 09) as a digital value via the A/D converter 08), where the calculation processing necessary for quantitative determination is performed. be exposed.

The intensity for each concentration was determined by subtracting the base from the peak of the waveform.

(2) Measuring device A measuring device using a flow type diluter according to the present invention shown in FIG. 1 was used. Each pump uses a delivery pump with a single plunger, and each pump has a different delivery rate than the first pump (
1) is 1.0ml/min, and the second pump (2) is 0.0ml/min.
9ml/min, third pump (3) 0.9ml/min
It was set to in.

The first carrier and the second carrier were common, and a 100 mM sodium phosphate buffer (pH 6.0) containing 1 mM sodium azide was used.

An autosampler was used as the sample injection section (4).

(3) Measurement method and results After the temperature in the thermostatic chamber 00 reached equilibrium, 5 μm of each 300 mM glucose aqueous solution was injected and measured, and the output current value per unit concentration was found to be 1.05 nA/mM.
Met. Furthermore, when using the apparatus of the present invention, high concentration samples could be measured in 1 minute per sample.

Comparative example 1 (1) Configuration of detection section The same material as in Example 1 was used.

(2) In measurement device Example 1, the extraction channel and the second pump (2)
, we used a device in which the confluence channel, third pump (3), diversion section (5), and confluence section (6) were removed, and the sample injection section (4) and detection section (7) were directly connected with a single pipe. there was. Further, the first pump (1 liter was set at 1.0ml/min).

(3) Measurement method and results Constant temperature chamber (after the +0 temperature reached equilibrium, 5 μm of 30 mM glucose aqueous solution was injected and measured, and the output current value per unit concentration was 10.92 n A / m
As a result, Example 1 was about 1064 compared to Comparative Example 1.
It turned out that it was diluted twice.

Example 2 (1) Configuration of detection section An alcohol detector schematically shown in FIG. 3 was used as the detection section (7). That is, using an alcohol oxidase immobilized column (21) and a hydrogen peroxide electrode (23), a flow cell (22) is placed in a constant temperature bath (20), and electrochemical detection is performed using a potentiostat (26). It was configured to detect alcohol. Details of the alcohol detector are shown below.

a. The side surface of a platinum wire with a diameter of 2 mm of hydrogen peroxide electrode was covered with heat-shrinkable Teflon.
Finish one end of the wire smooth with a file and 1500 emery paper. This platinum wire was used as a working electrode, a 1 cm square platinum plate was used as a counter electrode, and a saturated calomel electrode (hereinafter abbreviated as SCE) was used as a reference electrode in 0.1 M sulfuric acid at +2. Electrolytic treatment was performed for 10 minutes in OV. After that, after washing the platinum wire thoroughly with water,
After drying at 0 °C for 10 minutes and soaking in an anhydrous toluene solution of 10% T-aminopropyltriethoxysilane for 1 hour,
Washed.

Raw serum albumin (manufactured by Sigma, FraCtion
V) Dissolve 20 mg in 1 ml of distilled water, and add glutaraldehyde to 0.2%. Quickly place 5 μm of this mixed solution on the platinum wire prepared earlier, and
Dry cure for 15 minutes at C. Connect this to the hydrogen peroxide electrode (2
3).

b. Method for making an immobilized enzyme column Firebrick (60-80 mesh graded product) 150
mg was thoroughly dried, 1 ml of a 10% anhydrous toluene solution of T-aminopropyltriethoxysilane was added, and the mixture was left to stand for 1 hour. After thoroughly washing the silane coupling agent with toluene and methanol, it is dried at 120°C for 2 hours. After cooling, 5
Add 0.5 ml of % glutaraldehyde aqueous solution and leave at room temperature for 1 hour. This carrier is thoroughly washed with water. Finally the pH
Wash with 7°0 sodium phosphate buffer to remove as much buffer as possible.

Alcohol oxidase (
An enzyme solution prepared by diluting 50 μl of a liquid enzyme preparation derived from Pichia 1111 (manufactured by Sigma) 10 times with a sodium phosphate buffer solution at pH 7.0 is added, and the mixture is allowed to stand under water cooling for 1 hour. After standing, wash thoroughly with buffer. This enzyme-immobilized carrier has an outer diameter of 3 mm, an inner diameter of 2 mm, and a length of 10 cm.
The alcohol oxidase-immobilized column (21) was prepared by filling the alcohol oxidase into a polytetrafluoroethylene resin tube.

(2) Measuring device A measuring device using a flow type diluter according to the present invention shown in FIG. 1 was used. Each pump uses a delivery pump with a single plunger, and each pump has a different delivery rate than the first pump (
1) is 1.4 ml/min, and the second pump (2) is 1.4 ml/min.
3ml/min, third pump (3) 0.9ml/min
It was set to in.

The first carrier and the second carrier were common, and 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM sodium azide was used.

An autosampler was used for the sample injection section (4).

(3) Measurement method and results After the constant temperature bath (20) temperature reaches equilibrium, 5.0.10.
0.15.0.20.0 (v/v)% ethyl alcohol aqueous solution, 5μ each! Calibration was performed by injecting and measuring in 1 minute per sample. The calibration curve at this time is shown in FIG. At this time, the output current value per unit concentration was found to be 14.57 nA/(v/v)%. 5.0
A linear relationship was obtained for samples up to 15.0 (V/V)%. However, 20. The concentration of O(v/v)% was outside the linear range.

Comparative example 2 (1) Configuration of detection section The same material as in Example 2 was used.

(2) In the measurement device Example 2, the second pump (2), the third pump (3), the dividing section (5), and the confluence section (6) are removed, and the sample injection device (4) and the detection section (7) are removed. ) were connected directly with a single pipe. Also, the first pump (1) is 1.0ml/m
It was set to in.

(3) Measurement method and results The output current measured by this alcohol detector is because the hydrogen peroxide produced by the immobilized enzyme column is dependent on the oxygen dissolved in the solution. There is a limit to the range in which the line represents a straight line. 0゜1~2.0 (v/v)%
When the upper limit of the linear range of the output current value was confirmed using a dilute aqueous ethyl alcohol solution, it was found to be approximately 380 nA. Without using the diluter of the present invention, approximately 1
．． Samples with a concentration higher than 8% cannot be measured.

In addition, when the output current value per unit concentration was calculated, 209
．． It was 8 nA/(v/v)%.

As a result, it was found that Example 2 was diluted by about 14°4 times as compared to Comparative Example 2.

Example 3 (1) Configuration of detection section The same material as in Example 2 was used.

(2) Measuring device The sample injection part (4 ) and the diverter (5), the same device as in Example 2 was used, except that it was inserted between the flow divider (5). The flow rate of each pump is
Same as Example 2.

(3) Measurement method and results After the constant temperature chamber temperature reaches equilibrium, 5.0110°0, ■5
．． Calibration was performed by injecting 5 μl of each 0.20.0 (v/v)% ethyl alcohol aqueous solution and performing calibration, which is shown in FIG.

In this case, unlike in Example 2, it was confirmed that ethyl alcohol at a high concentration of 20.0 (v/v)% also fell on the calibration curve with high accuracy. This is thought to be due to insufficient mixing of the high concentration ethyl alcohol solution and buffer in Example 2, which did not show linearity at high concentrations.
In this example, it is considered that mixing was effectively promoted by the mixing piping provided.

Note that the output current value per unit concentration at this time is 13.
It was 22 nA/(v/v)%.

(Effects) The flow type diluter of the present invention does not require a needle for separating and dispensing a sample related to dilution and a drive mechanism thereof, and can be configured with a simple mechanism.

Therefore, it does not take much time for cleaning, mixing, stirring, etc. in the dilution operation.

When the flow-type diluter of the present invention is used, dilution can be performed continuously and in a short time, and sample sampling, dilution, and detection can be performed continuously, allowing rapid and accurate measurement. Therefore, when measuring a highly concentrated sample, it can be diluted at an accurate magnification with a simple operation.

[Brief explanation of drawings]

FIG. 1 is a system diagram showing an example of a flow type measuring device constructed using the flow type diluter of the present invention. Note that the arrow in the figure indicates the direction in which the carrier is fed. (1) First pump (2) Second pump (3) Third pump (4) Sample injection section (5) Diversion section (6) Confluence section (7) Detection section (8) First carrier bottle (9) Second carrier bottle (10) Waste liquid bottle FIG. 2 is a schematic diagram of a glucose detector which is an example of a detection section. (11) Thermostat (12) Mixing coil (13) Flow cell (14) Immobilized enzyme electrode (15) Ag, /AgCI reference electrode (16) Counter electrode (17) Potentiostat (18) A/D converter (19) ) Computer FIG. 3 is a schematic diagram of an alcohol detector, which is an example of the detection section. (20) Constant temperature bath (21) Alcohol oxidase immobilization column (22)
Flow cell (23) Hydrogen peroxide electrode (24) Ag/Agcl reference electrode (25) Counter electrode (26) Potentiostat (27) A/D converter (28) Computer Figure 4 shows the ethyl alcohol aqueous solution in Example 2. The calibration curve when measured as a sample is shown. FIG. 5 shows a calibration curve measured in Example 3 using an aqueous ethyl alcohol solution as a sample.

Claims (3)

[Claims] (1) a first pump for feeding the first carrier;
This is a flow type diluter that has a sample injection part and a main flow path connecting the sample injection part and the detection part, and has a flow diversion part downstream of the sample injection part on the main flow path, and a liquid flow from the separation part. a second pump and a second pump for extracting a part of the carrier, and further includes a confluence section on the main flow path after the branch section, and a confluence section for sending a second carrier to the confluence section. A flow type diluter characterized by having a flow path and a third pump. (2) The flow type diluter according to claim (1), further comprising a mixing pipe that generates turbulent flow within the pipe between the sample injection part and the flow dividing part. (3) The flow type diluter according to claim (2), wherein the mixing pipe is a pipe filled with granular material.