JP5386927B2 - Micro flow rate liquid feeding device and liquid feeding method - Google Patents

Description

  The present invention relates to an apparatus and a method for flowing a minute flow rate of several hundred nL to several tens of μL per minute.

  Although there are devices that can flow a minute flow rate of several hundred nL to several tens of μL per minute, they have many drawbacks. A pump having a stroke mechanism well known in liquid chromatography cannot be expected to stably flow the minute flow rate. Alternatively, the syringe-type pump can flow relatively stably even at the minute flow rate, but the syringe capacity is limited, and continuous use is difficult and differences between batches are likely to occur. There are challenges.

  A split method (static split method) is known as a classic method for flowing a minute flow rate. A flow rate of several tens to several hundred times the target flow rate is flowed and branched using the branching means (13) in front of the separation medium (7). This is a method of flowing to the side (A side flow path) and flowing most of the remainder to the disposal side (B side flow path) (see FIG. 1). In this method, the branching ratio is determined by the pressure ratio of the A side channel and the B side channel. In a steady state, a minute flow rate can be flowed, but when the pressure ratio between the A-side channel and the B-side channel changes due to a disturbance, there is a disadvantage that the branching ratio changes and the target minute flow rate also changes ( Patent Documents 1 and 2).

  The active split method has been proposed to solve the drawbacks of the static split method (Patent Document 3). In this method (see FIG. 2), a thermal flow meter (5a) is disposed in the A side flow path or the B side flow path, and a movable orifice valve (flow rate control device) (5b) is used so that the flow rate is constant. It is something to control. Since a device for detecting and capturing the target component is often arranged on the downstream side of the separation medium (7), a thermal flow meter (5a) is arranged in the B-side channel as in the apparatus of FIG. It is common. This method has less influence on the minute flow rate flowing through the A-side flow path than the static split method, even if disturbance due to physical separation of the separation medium or each pipe occurs. However, the active split method also has some problems.

  As a first problem, control is performed to keep the flow rate of the B-side flow path constant. Therefore, when the flow rate before branching changes, all the changes are flow rate changes of the A-side flow path, which is the target flow path.

For example, in the case of the apparatus shown in FIG. 2, the flow rate value (F _b ) of the B-side channel is measured at an arbitrary sampling interval by the thermal flow meter (5a), and the flow rate control device (5b) is set so that the value becomes constant. To control. The difference between this value and the flow rate before branching (F _m ) is sent to the A-side flow path (F _a ). The flow rate change is shown in FIG.

F _a = F _m −F _b
F _a : A side flow rate (micro flow rate)
F _b : Flow rate value of B side channel (large flow rate)
F _m : Flow before branching Since the thermal flow meter (5a) used here is highly accurate, even if the flow (F _m ) before branching fluctuates, the B-side flow path can be kept constant. (FIG. 3A). Therefore, when the flow rate (F _m ) before branching varies, most of the deviation affects the flow rate (F _a ) of the A-side flow path (FIG. 3B). For example, if the flow rate before branching (F _m ) is 50 μL / min and the flow rate (F _b ) of the B-side channel is 49 μL / min, the flow rate (F _a ) of the A-side channel is theoretically 1 μL per minute. When the flow rate before branching (F _m ) fluctuates ± 0.2 μL per minute, the flow rate (F _b ) of the B side channel is controlled at 49 μL per minute, so the flow rate (F _a ) of the A side channel is It fluctuates from 0.8 to 1.2 μL per minute, resulting in an error of ± 20%.

  As a second problem, when the specific heat of the flowing liquid changes (a method such as a solvent gradient used in liquid chromatography), the response of the thermal flow meter changes. For example, when acetonitrile is used in a thermal flow meter that has been calibrated with water, the response output is about 30% that of water even though the flow rate is the same. Therefore, in the method of flowing a minute flow rate by the active split method using the thermal type flow meter, when acetonitrile is flowed after water, the response output of the thermal type flow meter is reduced if the liquid feeding amount is constant. In order to make the response output constant, it is necessary to increase the flow rate of the B-side flow path, and as a result, the flow rate of the A-side flow path decreases. Therefore, it becomes more difficult to flow a minute flow rate by the active split method.

JP 2004-309135 A JP-T-2004-506896 JP 2006-276021 A

  It is an object of the present invention to provide a minute flow rate liquid feeding means and a liquid feeding method for flowing a minute flow rate of several hundred nL to several tens of μL per minute in the analyzer.

The present invention made in view of the above problems includes the following inventions:
According to the first aspect of the present invention, the liquid is flowed by the liquid feeding means, and the branching means is used to branch the flow into the separation medium side flow path and the waste side flow path. A liquid delivery device,
A first flow meter for measuring a flow rate value of the liquid flowing between the liquid feeding means and the branching means at an arbitrary sampling interval;
A second flow meter that measures the flow rate value of the liquid flowing in the separation medium side flow path or the waste side flow path at the same or different sampling interval as the first flow meter;
A flow rate control means for controlling the flow rate of the liquid flowing in the flow path on the separation medium side or the flow path on the waste side as necessary;
Based on the flow rate values measured by the first and second flow meters, the flow rate of the liquid flowing in the flow path between the liquid feeding means and the branching means, the flow path on the separation medium side, and the flow path on the disposal side is calculated. Arithmetic means for sending a signal for control,
The calculation means (1-1) uses the flow value measured by the first flow meter as an input value, multiplies the flow value by an arbitrary value less than 1, and the flow rate to be measured by the second flow meter. Calculate the value
(1-2) Using the flow rate value measured by the second flow meter as an input value, comparing it with the calculated flow rate value, and controlling the flow rate control means so as to match both flow rate values;
Or
(2-1) Using the flow value measured by the second flow meter as an input value, multiply the flow value by an arbitrary value exceeding 1, and calculate the flow value to be measured by the first flow meter. ,
(2-2) Using the flow rate value measured by the first flow meter as an input value, comparing it with the calculated flow rate value, and controlling the liquid feeding means so as to match both flow rate values;
The device is characterized by the above.

  A second invention is the micro flow rate liquid feeding device according to the first invention, wherein the first and second flow meters are thermal flow meters.

  The third invention is the micro flow rate liquid feeding device according to the first to second inventions, characterized in that the second flow meter and the flow rate control means are installed in the flow path on the disposal side. is there.

According to a fourth aspect of the present invention, the calculation means includes a time measured at each sampling interval.
(1) Flow rate values measured by the first and second flow meters,
(2) a calculated value of the flow rate to be measured by the first or second flow meter,
(3) Flow rate value to be controlled by the flow rate control means or the liquid feeding means,
(4) Information relating to control of the flow rate control means or the liquid feeding means,
The micro flow rate liquid feeding device according to any one of the first to third inventions, comprising a recording means for recording

According to a fifth aspect of the present invention, a liquid is flowed using a liquid feeding means, and is branched into a flow path on the separation medium side and a flow path on the disposal side using a branching means, so that a minute flow rate of liquid flows through the separation medium. A flow rate feeding method,
Measure the flow rate value of the liquid flowing between the liquid feeding means and the branching means and the flow rate value of the liquid flowing in the separation medium side flow path or the disposal side flow path,
(1-1) The flow rate value of the liquid flowing between the liquid feeding means and the branching means is multiplied by an arbitrary value less than 1, and the liquid to be flowed to the separation medium side flow path or the waste side flow path Calculate the flow value,
(1-2) The calculated flow rate value and the flow rate value of the liquid flowing in the separation medium side flow path or the disposal side flow path are matched with the separation medium side flow path or the waste side flow path. Control the flow rate of flowing liquid,
Or (2-1) multiplying the flow rate of the liquid flowing in the flow path on the separation medium side or the flow path on the waste side by an arbitrary value greater than 1, and the liquid to flow between the liquid feeding means and the branching means Calculate the flow value,
(2-2) The flow rate of the liquid flowing between the liquid feeding unit and the branching unit is controlled so that the calculated flow rate value matches the flow rate value of the liquid flowing between the liquid feeding unit and the branching unit. To
The method is characterized by the above.

  According to a sixth aspect of the present invention, the flow rate controlled by a calculated value obtained by multiplying the flow rate value of the liquid flowing between the liquid feeding unit and the branching unit by an arbitrary value less than 1 is the flow rate of the liquid flowing in the waste-side flow path. The micro flow rate liquid feeding method according to the fifth aspect of the present invention.

  Hereinafter, the present invention will be described in detail.

The present invention measures the flow rate value of the liquid flowing between the liquid feeding means and the branching means, and the flow rate value of the liquid flowing in the separation medium side flow path or the waste side flow path,
The flow rate value obtained by multiplying the flow rate value of the liquid flowing between the measured liquid feeding means and the branching unit by an arbitrary flow rate ratio of less than 1 is calculated, and the calculated flow rate value and the flow path on the separation medium side or the waste side The flow rate of the liquid flowing in the separation medium side flow path or the disposal side flow path is controlled so that the measured value of the flow rate of the liquid flowing in the flow path matches.
Or
The flow rate value obtained by multiplying the measured flow rate value of the liquid flowing in the separation medium side flow path or the waste side flow path by an arbitrary flow rate ratio exceeding 1 is calculated, and the calculated flow rate value, the liquid feeding means, and the branching means By controlling the flow rate of the liquid flowing between the liquid feeding unit and the branching unit so that the measured value of the flow rate of the liquid flowing between
It is characterized by flowing a liquid with a constant minute flow rate into the flow channel on the separation medium side.

  The method for measuring the flow rate value in the micro flow rate feeding method of the present invention is not limited as long as an apparatus capable of measuring the liquid flow rate is used, but in particular, a thermal flow meter capable of measuring a micro flow rate liquid with high accuracy. It is preferable to measure using

  In the minute flow rate liquid feeding method of the present invention, when controlling the flow rate of the liquid flowing in the separation medium side flow path or the disposal side flow path, the flow rate of the liquid flowing in the same flow path as the flow path for measuring the liquid flow rate (For example, if the flow channel for measuring the flow rate is a flow channel on the separation medium side, the flow rate of the flow channel on the separation medium side may be controlled), or a flow channel different from the flow channel measuring the liquid flow rate The flow rate of the liquid flowing through the flow path may be controlled (for example, if the flow rate measurement channel is a separation medium side flow rate, the flow rate of the waste side flow channel may be controlled). Is preferred.

Examples of the flow rate control method for the liquid flowing in the separation medium side flow path or the waste side flow path using the flow rate control means in the micro flow rate liquid feeding method of the present invention include the following methods.
[1] A method of controlling a flow rate by installing a valve on a flow path for controlling the flow rate.
[2] A method of controlling the flow rate by installing a valve in the branching means.
Among these, as the valve in the method [1] or [2], a gate valve (gate valve), a globe valve (ball valve), a butterfly valve, and a diaphragm valve that are usually used for flow rate control can be exemplified. It is preferable to use a valve using an orifice plate capable of controlling a minute flow rate. In the flow control using a valve using an orifice plate,
[3] Control that starts from a state where the orifice plate is fully opened, and narrows the orifice plate from that state.
[4] Control in which the orifice plate starts control from the intermediate opening state, and the orifice plate is expanded or narrowed from that state.
Any control may be performed. In the control [3], when the flow rate is increased from the initial flow rate, it is necessary to control the flow rate of the liquid flowing between the liquid feeding means and the branching means. The range can be increased compared to the control in [4]. On the other hand, although the range of the flow rate that can be controlled by the orifice plate is narrower than the control of [3], the control of [4] can be performed by expanding the orifice plate even when the flow rate is increased from the initial flow rate. it can.

  Examples of the flow rate control method for the liquid flowing between the liquid feeding unit and the branching unit in the micro flow rate liquid feeding method of the present invention include a method for controlling the flow rate of the liquid flowing through the liquid feeding unit.

The minute flow rate control method of the present invention may be a method of performing control of either [1] or [2] below, or a method of switching control of [1] and [2] as necessary. Good.
[1] Controlling the flow rate of the liquid flowing in the separation medium side channel or the disposal side channel [2] Controlling the flow rate of the liquid flowing in the channel between the liquid feeding means and the branching means The latter minute flow rate control method Is used, there is a large difference between the measured flow rate value of the liquid flowing in the separation medium side or waste liquid side flow path and the calculated flow rate value, and the flow rate installed in the separation medium side or waste liquid side flow path. Even when the control device cannot control the flow rate (for example, when the orifice valve is fully opened), it switches to the flow rate control of the liquid flowing by the liquid feeding means, and flows to the flow path on the separation medium side or the waste liquid side. The liquid flow rate can be controlled.

  The separation medium installed in the separation medium side flow path is not particularly limited as long as it meets the purpose of analyzing / sorting a micro sample. Examples include electrophoresis columns, analysis microplates such as electrophoresis, and fractionation microplates. Moreover, the liquid feeding method of the present invention can also be adopted as a constituent element of a microreactor in which a collected sample is reacted with another reagent.

  FIG. 4 shows an embodiment of a liquid feeding device using the micro flow rate liquid feeding method of the present invention. The eluent (1) is fed at a constant flow rate by the liquid feed pump (2). The sent eluent (1) passes through the first thermal flow meter (4), and then is separated by the branch block (13) on the separation medium side (hereinafter referred to as A side flow path) and the waste side (hereinafter referred to as (B side channel). A second thermal flow meter (5a), a flow rate control device (5b), a sample injection device (6), an analysis column (7), and a detector (9) are arranged in the flow path of the A side in the order in which the liquid flows. ing. The liquid flowing in the B side flow path becomes the waste liquid (11) as it is. The first thermal flow meter (4) and the second thermal flow meter (5a) are installed close to each other. Further, although not shown in FIG. 4, pressure adjusting means such as a resistance tube may be installed in the B side channel as necessary.

The flow rate value of the eluent (1) sent by the pump (2) is changed by the first thermal flow meter (4), and the flow rate value of the eluent flowing in the A side flow path is changed by the second thermal flow meter ( 5a), each is measured at an arbitrary sampling interval, and the value is input to the calculator (14), and then the calculator (14) uses the first thermal flow meter (4) among the measured flow values. Multiply the flow rate value measured in step 1 by an arbitrary flow rate ratio less than 1 to calculate the flow rate that should flow through the A-side flow path. Then, the calculated flow rate value is compared with the flow rate value measured by the second thermal flow meter (5a), and a signal for controlling the flow rate of the flow rate control device (5b) so that both flow rate values coincide with each other. By sending from the calculator (14), the flow rate of the eluent flowing in the A-side flow path is controlled. Thereby, a minute flow rate of several hundred nL to several tens of μL per minute flows in the A-side flow channel, and a difference between the flow rate before branching and the flow rate of the A-side flow channel flows in the B-side flow channel. The calculator (14) may exist alone or may be included in the first thermal flow meter (4) or the second thermal flow meter (5a). The calculator (14) is provided with a program for performing the control, and (1) a flow rate value measured by the first and second thermal flow meters,
(2) Calculated value of the flow rate to be measured by the second thermal flow meter,
(3) The flow value to be controlled by the flow control device,
(4) Information relating to control of the flow control device,
It is preferable that a recording means for recording the above is provided from the viewpoint of efficiently performing the control.

  In the liquid sending means shown in FIG. 4 in which the second thermal flow meter (5a) is installed in the A side flow path, the second capacity having an internal capacity of at least about several hundred μL before the analytical column (7). A thermal flow meter (5a) and a flow control device (5b) are installed. Since the internal capacity of the second thermal flow meter (5a) and the flow control device (5b) is large with respect to the A-side flow path through which a minute flow rate of several hundreds nL to several tens of μL per minute, When a very small flow rate of 100 nL is allowed to flow through the A-side flow path, the eluent flowing through the first thermal flow meter (4) reaches the analysis column (7) installed at the outlet of the flow control device (5b). It takes a considerable amount of time (if the flow rate of the A side channel is 500 nL / min and the internal volume of the flow meter and the control device is 500 μL, the eluent passes through the flow meter and the control device. Takes 1000 minutes for calculation), and there is a delay in the time to reach the analytical column (7).

  In the case of a system in which the composition of the liquid flowing in the separation medium does not change (for example, elution by isocratic method, column washing, buffer replacement), the analysis column (7 The problem that the time to reach) is delayed can be solved. Further, since the first thermal flow meter (4) and the second thermal flow meter (5a) are installed close to each other, a delay in time to reach the second thermal flow meter can be ignored.

  On the other hand, in a system in which the liquid composition changes over time (for example, elution by the solvent gradient method), the delay in reaching the separation column directly leads to a delay in response to the composition change. Even if the flow rate is controlled by the flow meter (5a) and the flow rate control device (5b), it is difficult to send an eluent having a desired flow rate and composition to the analytical column (7).

  FIG. 5 shows another embodiment of the liquid feeding device using the micro flow rate liquid feeding method of the present invention.

  4 is different from the apparatus of FIG. 4 in that the arrangement of the A-side flow path is in the order of flow of the liquid, the sample injection device (6), the analysis column (7), the detector (9), and the second thermal flow meter (5a). The flow control device (5b) is provided. Although not shown in FIG. 5, a pressure adjusting means such as a resistance tube may be installed in the B side flow path as necessary. The apparatus of FIG. 5 can also produce a minute flow rate of several hundred nL to several tens of μL per minute in the A-side flow channel, as in FIG.

  In the case of a system in which the composition of the liquid flowing in the separation medium does not change (for example, elution by isocratic method, column washing, buffer solution replacement), the eluent is flowed to the liquid feeding means in advance as in the apparatus of FIG. Thus, the problem that the time to reach the separation column is delayed can be solved. An analytical column (7) having an internal capacity is installed between the first thermal flow meter (4) and the second thermal flow meter (5a). By flowing the eluent, the problem of delaying the time to reach the second thermal flow meter can be solved.

  In a system in which the composition of the liquid changes over time (for example, elution by the solvent gradient method), as in the case of FIG. 4, the delay in the arrival time to the analytical column (7) remains as a delay in the response to the composition change. However, when the first thermal flow meter (4) and the analytical column (7) are installed close to each other, the problem of delaying the time to reach the analytical column (7) can be ignored. However, since an analytical column (7) having an internal capacity is installed between the first thermal flow meter (4) and the second thermal flow meter (5a), the second thermal type The problem that the time to reach the flow meter is delayed, that is, the problem that the response to the composition change is delayed cannot be ignored. In addition, when another analysis device such as a fractionation (collection) device for the separated target component and a mass spectrometer is installed after the second thermal flow meter (5a) and the flow control device (5b), the second The thermal flow meter (5a) and the flow control device (5b) have an internal capacity of at least several hundreds μL, so that an eluent having a desired flow rate and composition can be supplied to the preparative device and other analysis devices. It becomes even more difficult to send the solution. In addition, when a fractionation (collection) device and other analysis devices are installed between the analysis column (7) and the second thermal flow meter (5a), the separation device and the other analysis device are reached. However, the distance between the first thermal flow meter (4) and the second thermal flow meter (5a) is further away, so the second thermal flow meter is reached. An even greater delay occurs in the time to do.

  FIG. 6 shows a preferred embodiment of a liquid feeding device using the micro flow rate liquid feeding method of the present invention.

  The difference from the apparatus of FIG. 4 is that the second thermal flow meter (5a) and the flow rate control device (5b) are arranged in the B-side flow path, and sample injection into the A-side flow path in the order of liquid flow. The device (6), the analytical column (7) and the detector (9) are arranged. The first thermal flow meter (4) and the second thermal flow meter (5a) are installed close to each other. Further, although not shown in FIG. 6, pressure adjusting means such as a resistance tube may be provided in the B side flow path as necessary.

  The flow rate value of the eluent (1) sent by the pump (2) is changed by the first thermal flow meter (4), and the flow rate value of the eluent flowing in the B side channel is changed by the second thermal flow meter ( 5a), each value is measured in real time, and the value is input to the calculator (14), and then measured by the calculator (14) with the first thermal flow meter (5a) among the measured flow values. The flow rate to be passed through the B-side flow path is calculated by multiplying the flow rate value by an arbitrary flow rate ratio of less than 1. Then, the calculated flow rate value is compared with the flow rate value measured by the second thermal flow meter (5a), and a signal for controlling the flow rate of the flow rate control device (5b) so that both flow rate values coincide with each other. By sending from the calculator (14), the flow rate of the eluent flowing in the B-side channel is controlled in real time. Thereby, the difference of the flow volume before a branch and the flow volume of a B side flow path flows into the A side flow path.

  In the case of the minute flow rate liquid delivery device of FIG. 6, a device having an internal capacity of at least about several hundred μL such as the second thermal flow meter (5a) and the flow rate control device (5b) Side channel). Therefore, the problem that the time to reach the analysis column (7) installed in the A-side flow path is delayed can be solved. In addition, since the first thermal flow meter (4) and the second thermal flow meter (5a) are installed close to each other, the time to reach the second thermal flow meter (5a) is delayed. Can also be eliminated. Furthermore, even in a system in which the composition of the liquid changes with time (for example, elution by the solvent gradient method), the second thermal flow meter (5a) and the flow control device ( Since there is no difference in the internal capacity of 5b), the problem of delay in the composition change of the eluent can be solved. Therefore, regardless of the system in which the composition of the liquid does not change with time, an eluent having a desired flow rate and composition can be passed through the analysis column (7).

In the liquid feeding method in the micro flow rate liquid feeding device of FIG. 6, the flow rate value before branching (flow rate value of the liquid flowing between the liquid feeding unit and the branching unit) (F _m ) is measured at an arbitrary sampling interval. Control is performed by multiplying the flow rate value by a certain flow rate ratio to obtain a flow rate control value for the B-side flow path. The flow rate change is shown in FIG.

F _b = F _m · S
F _a = F _m −F _b
F _a : A side flow rate (micro flow rate)
F _b : Flow rate value of B side channel (large flow rate)
F _m : Flow rate value before branching S: Flow rate ratio In the liquid feeding method in the minute flow rate liquid feeding means in FIG. 6, the first thermal equation is used so that the flow rate value (F _m ) before branching can be measured at an arbitrary sampling interval. A flow meter (4) is installed. The flow rate value (F _m ) before branching is measured at an arbitrary sampling interval, and the value is multiplied by a constant flow rate ratio (S) of less than 1 to give a control flow rate value for the B-side flow path. Therefore, even when the flow rate value (F _m ) before branching varies, the flow rate value (F _b ) of the B-side flow path is multiplied by the flow rate ratio (S) to the flow rate value (F _m ) before branching. The flow rate (F _a ) of the A-side channel is the difference between the values (FIG. 7 (a)). Therefore, the fluctuation of the flow rate (F _a ) of the A-side flow path is smaller than that of the minute flow rate liquid feeding method by the conventional active split method (FIG. 3B) controlled by the absolute value of the flow rate (FIG. 7 ( b)). For example, in the apparatus shown in FIG. 6, when the flow rate before branching (F _m ) is 50 μL / min and the branching ratio (S) is 0.98, the calculated flow rate value (F _b ) of the B side channel is The flow rate (F _a ) of 49 μL and the A side channel is 1 μL per minute. When the flow rate value (F _m ) before branching varies by ± 0.2 μL per minute, the flow rate value (F _b ) of the B-side flow channel varies from 48.80 to 49.12 μL per minute. The flow rate (F _a ) of the A-side flow path varies from 0.996 to 1.004 μL per minute and falls within an error of 0.008 μL (± 0.4%) per minute. On the other hand, under the same conditions (flow before branching: 50 μL per minute, B side flow rate: 49 μL per minute, flow rate fluctuation: ± 0.2 μL per minute), minute flow rate by the conventional active split method (FIG. 2) In the liquid feeding method, the flow rate fluctuation of the A-side channel is 0.4 μL (± 20%) per minute.

  In the case of a solvent gradient elution method in which the composition of the eluent changes with time, it is more difficult to flow a minute flow rate by the conventional active split method.

  Since the thermal flow meter is corrected in advance with the eluent used, when a different eluent is flowed, the value deviates from the actual value. The output of the thermal flow meter varies depending on the specific heat of the eluent used as shown in the following equation.

V _signal = k · C _p · Φ
V _signal = Output flow rate value k = Coefficient of correction C _p = Specific heat Φ = Actual flow rate In FIG. 8, a thermal flow meter corrected with water was used, and a gradient from 100% water to 100% acetonitrile was performed. Shows the change in the output of the thermal flow meter. The specific heat of water is 1.00 Cal / (g · deg), and the specific heat of acetonitrile is 0.304 Cal / (g · deg). When a thermal flow meter corrected with water is used, if the water is fed at 1.00 mL per minute, the thermal flow meter has an output value of 1.00 mL per minute. However, when acetonitrile is fed as an eluent at 1.00 mL / min, the thermal flow meter has an output value of 0.304 mL / min. In other words, when a gradient from 100% water to 100% acetonitrile is performed by the solvent gradient elution method, the output of the thermal flow meter decreases with time even though the actual flow rate is constant. Due to such characteristics, there is a problem with the conventional micro flow rate liquid feeding method by the active split method. In the solvent gradient elution method, the minute flow rate liquid feeding method by the conventional active split method (FIG. 2) and the present invention shown in FIG. The difference from the minute flow rate liquid feeding method will be described.

First, the conventional micro flow rate feeding method using the active split method is as follows (see FIG. 9). When the flow rate before branching (F _m ) is 50 μL per minute and the flow rate (F _b ) of the B side channel is 49 μL per minute, the flow rate (F _a ) of the A side channel is initially 100% water. Is 1 μL of the difference per minute (FIG. 9A). As the proportion of acetonitrile gradually increases, the actual flow rate in the B-side channel hardly changes, but the response of the thermal flow meter decreases. It is controlled and keeps a constant value (49 μL / min) (FIG. 9A). With acetonitrile 100%, the value of the target control flow rate for the B-side channel increases by 229%, which is calculated to be 161 μL per minute. The flow rate (F _a ) of the A side channel becomes −112 μL per minute of the difference, and cannot be controlled (FIG. 9B).

  In the minute flow rate liquid feeding method of the present invention shown in FIG. 6, under the same conditions, the following is performed (see FIG. 10).

When the flow rate before branching (F _m ) is controlled at 50 μL / min and the flow rate ratio to the B-side flow path is 0.98, the water flow is initially 100%, so the calculated flow rate value of the B-side flow path (F _b ) Is 49 μL / min, obtained by multiplying the flow rate before branching (F _m ) by 50 μL / min by 0.98 (FIG. 10 (a)), and the flow rate (F _a ) of the A side channel is the difference per minute 1 μL. As the proportion of acetonitrile gradually increases, the response of the first thermal flow meter and the second thermal flow meter decrease at the same rate even though the actual flow rate hardly changes ( FIG. 10 (a)). With 100% acetonitrile, the output flow value of the first thermal flow meter before branching is 15 μL / min (50 × 0.30), and the target control flow rate value for the B-side channel is calculated to be the flow rate value before branching. (F _m ) 14.7 μL / min, obtained by multiplying 15 μL / min by 0.98. The flow rate (F _a ) of the A-side flow path is calculated to be 0.3 μL per minute of the difference (FIG. 10B). However, in reality, although the output value of the thermal flow meter is reduced by 70%, the actual flow rate is hardly changed, so the flow rate (F _b ) of the B side channel is the flow rate before branching even with 100% acetonitrile. (F _m ) 49 μL per minute obtained by multiplying 50 μL per minute by 0.98, and the flow rate (F _a ) of the A-side channel is 1 μL per minute of the difference.

  In addition, in the micro flow rate liquid feeding method of FIG. 6, as the flow rate ratio (S) increases (closer to 1), the flow rate error has an advantage over the micro flow rate liquid feeding method by the conventional active split method (FIG. 2). The branching ratio is preferably 0.7 (A-side flow path: B-side flow path = 30: 70) to 0.999 (A-side flow path: B-side flow path = 1: 1000).

The minute flow rate liquid feeding method of the present invention is:
Measure the flow rate value of the liquid flowing between the liquid feeding means and the branching means and the flow rate value of the liquid flowing in the separation medium side flow path or the disposal side flow path,
(1-1) The flow rate value of the liquid flowing between the liquid feeding means and the branching means is multiplied by an arbitrary value less than 1, and the liquid to be flowed to the separation medium side flow path or the waste side flow path Calculate the flow value,
(1-2) The calculated flow rate value and the flow rate value of the liquid flowing in the separation medium side flow path or the disposal side flow path are matched with the separation medium side flow path or the waste side flow path. Control the flow rate of flowing liquid,
Or
(2-1) Multiplying the flow rate value of the liquid flowing through the separation medium side flow path or the disposal side flow path by an arbitrary value exceeding 1, and the amount of liquid to flow between the liquid feeding means and the branching means. Calculate the flow value,
(2-2) The flow rate of the liquid flowing between the liquid feeding unit and the branching unit is controlled so that the calculated flow rate value matches the flow rate value of the liquid flowing between the liquid feeding unit and the branching unit. To
Thus, a constant minute flow rate is caused to flow through the separation medium. For this reason, even when the flow rate of the liquid flowing between the liquid feeding means and the branching means fluctuates due to a problem on the liquid feeding means side (for example, pulsation of the pump), the flow path on the separation medium side or the waste side flow path Compared to the conventional active split method, which controls only the absolute value of the flow rate of the flowing liquid, the flow rate fluctuation can be suppressed, so that a liquid with a constant minute flow rate can be accurately fed to the flow path on the separation medium side. Can do.

  Since the minute flow rate liquid feeding method of the present invention is controlled by the flow rate ratio, the smaller the flow rate flowing to the flow path on the separation medium side, the more accurate the flow rate accuracy compared to the conventional active split method that controls by the absolute value of the flow rate. Superiority. In particular, the ratio of the flow rate of the liquid flowing in the flow path on the disposal side to the flow rate of the liquid flowing between the liquid feeding means and the branching unit is a value close to 1, specifically 0.7 (separation medium side: disposal side) = 30: 70) to 0.999 (separation medium side: discard side = 1: 1000), the advantage over the conventional active split method is enhanced.

An apparatus adopting the micro flow rate liquid feeding method of the present invention, specifically, a first flow meter for measuring a flow rate value of a liquid flowing between the liquid feeding means and the separation means at an arbitrary sampling interval, and a separation medium A second flow meter that measures the flow rate of the liquid flowing in the flow channel on the side or the waste flow channel at the same or different sampling interval as the first flow meter, and the flow channel on the separation medium or the waste flow channel And a flow path between the liquid feeding means and the branching means based on the flow rate values measured by the first and second flow meters and the flow rate control means for controlling the flow path of the liquid flowing through And a calculation means for sending a signal for controlling the flow rate of the liquid flowing in the medium side flow path and the waste side flow path, and the calculation means is measured by (3-1) the first flow meter. The second flow rate is obtained by multiplying the flow rate value by any value less than 1 In calculates the flow rate value to be measured,
(3-2) Using the flow rate value measured by the second flow meter as an input value, comparing it with the calculated flow rate value, and controlling the flow rate control means so as to match both flow rate values;
Or (4-1) Using the flow value measured by the second flow meter as an input value, multiplying the flow value by an arbitrary value exceeding 1, and calculating the flow value to be measured by the first flow meter And
(4-2) The flow rate value measured by the first flow meter is used as an input value, compared with the calculated flow rate value, and the liquid feeding means is controlled to match both flow rate values.
Even if the specific heat fluctuates with the composition fluctuation of the liquid flowing in the flow path, and the flow value indicated by the thermal flow meter fluctuates accordingly, the micro flow rate liquid feeding device is a means to the flow path on the separation medium side. Since the flow rate is controlled by the flow rate ratio, the flow rate of the liquid flowing in the separation medium side channel or the waste side channel is controlled by the absolute value of the flow rate indicated by the thermal flow meter. In comparison, since the actual flow rate fluctuation can be suppressed, a liquid with a constant minute flow rate can continue to flow through the flow path on the separation medium side with high accuracy.

  In the apparatus, the composition of the liquid flowing in the separation medium changes even if the second thermal flow meter and the flow control device are installed in any of the separation medium side channel and the disposal side channel. In systems that do not (for example, elution by isocratic method, column washing, and buffer solution replacement), it is possible to provide a liquid delivery device that is superior in micro flow rate control as compared with the conventional active split method.

  Further, in the apparatus, analysis in a system (for example, a solvent gradient system) in which the composition of the liquid flowing in the separation medium changes with time, fractionation (collection) of target components separated in the analysis, and other analysis apparatuses When the second thermal flow meter and the flow control device are installed on the inlet side of the separation medium and the sorting / analyzing device, the flow rate ( The internal capacity (several hundred μL) of the second thermal flow meter and the flow rate control device with respect to several hundreds nL to several tens μL per minute is large, and a large delay occurs in the liquid composition or the sample arrival time. Therefore, when the present invention is applied to the analysis, the second thermal flow meter and the flow control device are installed in the separation medium and the flow path on the outlet side or the disposal side of the sorting / analyzing device. Is preferred.

  In particular, when both the second thermal flow meter and the flow control device are installed in the flow path on the disposal side, the first thermal flow meter, the second thermal flow meter and the flow control device are installed close to each other. Analysis in a system in which there is no delay in response and the composition of the liquid flowing in the separation medium does not change, analysis in a system in which the composition of the liquid flowing in the separation medium changes over time, and the analysis Thus, it is possible to provide a device capable of controlling a minute flow rate in any analysis, ie, fractionation (collection) of the target component separated in (1) and analysis using another analysis device.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 Comparison of liquid feeding methods in isocratic elution method Conventional analysis in the case of performing analysis under conditions of flow rate of several tens of μl per minute by isocratic elution method without composition change of the liquid flowing in the thermal flow meter Comparison was made between the micro flow rate feeding method by the active split method and the micro flow rate feeding method of the present invention.

(A) Microflow feeding method by conventional active split method FIG. 2 shows a system configuration of a microflow feeding device by a conventional active split method. The eluent (1) is fed at a constant flow rate by the liquid feed pump (2), and the sent eluent (1) is branched into the A side channel and the B side channel by the branch block (13). The A-side channel is a channel used for analysis, and a sample injection device (6), an analysis column (7), a column thermostat (8), and a detector (9) are arranged. A thermal flow meter (5a) and a flow rate control device (5b) are arranged in the B side flow path. The A-side channel is a channel through which a minute flow rate of several hundreds nL to several tens of μL per minute flows. The B-side channel is a channel through which most of the rest flows.

  The measurement conditions were an ODS column (manufactured by GL Science) having an inner diameter of 0.3 mm, a length of 50 mm and a particle diameter of 3 μm as the analytical column (7), and acetonitrile / water (40/60) as the eluent (1). . As a pump (2), CCSO made by Tosoh Corporation was partially improved and used. The flow rate of the pump (2) is set to 50 μL / min and liquid feeding is performed, and the flow rate value of the B side channel is constant at 47.7 μL / min with the thermal flow meter (5a) and the flow rate control device (5b). Thus, the control was performed so that 2.3 μL per minute, which is the above difference, flows through the A-side flow channel for analysis.

  As a measurement sample, a mixture of four kinds of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and n-butyl p-hydroxybenzoate was used.

(B) Micro flow rate liquid feeding method of the present invention FIG. 6 shows the system configuration of the micro flow rate liquid feeding device of the present invention. The eluent (1) is fed at a constant flow rate by the liquid feed pump (2). The sent eluent (1) passes through the first thermal flow meter (4) and is then branched into the A-side channel and the B-side channel by the branch block (13). The A-side channel is a channel used for analysis, and a sample injection device (6), an analysis column (7), a column thermostat (8), and a detector (9) are arranged. A second thermal type flow meter (5a) and a flow rate control device (5b) are arranged in the B side flow path. The A-side channel is a channel through which a minute flow rate of several hundreds nL to several tens of μL per minute flows. The B-side channel is a channel through which most of the rest flows.

  The measurement conditions were an ODS column (manufactured by Tosoh Corporation) having an inner diameter of 0.3 mm, a length of 50 mm and a particle diameter of 3 μm as the analytical column (7), and acetonitrile / water (30/70) as the eluent. As a pump (2), CCSO made by Tosoh Corporation was partially improved and used. The flow rate of the pump (2) was set to 50 μL / min for liquid feeding. For the eluent, the flow rate value is measured by the first thermal flow meter (4), and a value obtained by multiplying the value (flow rate value) by the flow rate ratio (0.944) is calculated by the calculator (14). Then, a control signal for matching the calculated flow rate value with the flow rate value of the second thermal flow meter (5a) is sent from the computing unit (14) to the flow rate control device (5b), It is the above difference (the first thermal flow meter of the first thermal flow meter) so that the flow rate value becomes minute (flow value of the first thermal flow meter × 0.944) μL per minute. The flow rate was controlled to be 0.056) μL.

  As the measurement sample, the same four kinds of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and n-butyl p-hydroxybenzoate as in the apparatus (A) were mixed. A thing was used.

(C) Results FIG. 11 is a diagram showing a signal from the thermal flow meter (5a) arranged in the B-side flow path in the system configuration (A) (FIG. 2). As shown in FIG. 11, the signal of the thermal flow meter (5a) is very stable, and it can be seen that the flow rate of the B-side flow path is accurately maintained constant. The chromatogram (n = 20) in the state of FIG. 11 is shown in FIG. 12, and the results of summarizing the Cv (%) of the elution time of each peak are shown in FIG. Despite the fact that the flow rate in the B-side channel is kept exactly constant, the reproducibility of the elution time is as bad as 1 to 4% in Cv (%). This is because the actual liquid delivery amount of the liquid feed pump fluctuates on the order of ± several hundreds nL per minute due to the temperature and the influence of bubbles in the eluent. While the actual liquid delivery amount of the liquid feed pump (2) is fluctuating, the flow rate of the B side flow path is kept constant, so ± 100 nL per minute due to the liquid feed pump (2) This is because the fluctuation in the order of the amount is directly added to or subtracted from the actual flow rate of the A-side flow path used for the analysis, resulting in a large flow rate fluctuation.

  13A is a signal from the first thermal flow meter (4) in the system configuration of FIG. 6B (FIG. 6), and FIG. 13B is a signal from the second thermal flow meter (5a). Respectively. FIG. 13C shows a value obtained by subtracting the signal from the second thermal flow meter (5a) from the signal from the first thermal flow meter (4), that is, the flow rate change in the A-side flow path used for analysis. FIG.

  As shown in FIG. 13 (a), the signal of the first thermal flow meter (4) seems to be stable at first glance, but fluctuates on the order of several hundred nL per minute. Since the flow rate of the B side channel is controlled to be a value obtained by multiplying the flow rate value of the first thermal flow meter (4) by the branching ratio, the B side channel is synchronized with the fluctuation of the actual flow rate. The flow rate (FIG. 13B) also fluctuates on the order of ± several hundreds nL per minute, indicating that feedback control is being performed correctly. Therefore, the fluctuation of the flow rate before branching can be canceled, and the signal from the first thermal flow meter (4) (FIG. 13 (a)) to the signal from the second thermal flow meter (5a) (FIG. 13 ( The difference of b)) is very stable compared to the system configuration of FIG. (A) (FIG. 2) (FIG. 13C).

  The chromatogram (n = 20) in the state of FIG. 13 is shown in FIG. 14, and Cv (%) of the elution time of each peak is shown in FIG. 15 (b). By using the system configuration (B) (FIG. 6), it becomes possible to keep the actual flow rate of the A side flow path used for analysis constant, and as a result, the reproducibility of the elution time is Cv (%). From 0.5 to 1%, the system configuration (A) in FIG. 2 (FIG. 2) was significantly improved compared to that in FIG. 15 (a).

Example 2 Comparison of liquid feeding method in solvent gradient elution method In the solvent gradient elution method in which the composition of the liquid flowing in the thermal flow meter changes with time, analysis was performed under conditions of several tens of μL per minute. A comparison between a conventional micro flow rate feeding method by the active split method and a micro flow rate feeding method of the present invention is shown.

(A) Microflow feeding method by conventional active split method FIG. 16 shows a system configuration of a microflow feeding device by a conventional active split method. The eluent A (1) is sent by the liquid feed pump A (2), and the eluent B (15) is sent by the liquid feed pump B (16). Solvent gradient is performed by changing the flow rates of the liquid feed pump A (2) and the liquid feed pump B (16) (the sum of the flow rates of the liquid feed pump A (2) and the liquid feed pump B (16) is constant). The sent eluent (1, 15) is branched into the A-side flow path and the B-side flow path by the branch block (13). The A-side channel is a channel used for analysis, and a sample injection device (6), an analysis column (7), a column thermostat (8), and a detector (9) are arranged. A thermal flow meter (5a) and a flow rate control device (5b) are arranged in the B side flow path. The A-side channel is a channel through which a minute flow rate of several tens of μL per minute to several hundreds of nL per minute flows. The B-side channel is a channel through which most of the rest flows.

  The measurement conditions were an analytical column (7), an ODS column (manufactured by Tosoh Corporation) having an inner diameter of 1 mm, a length of 50 mm and a particle size of 3 μm, and an eluent (1, 15) as A: acetonitrile / water (20/80), B : Acetonitrile / water (70/30) was used. As a pump (2, 16), CCSO manufactured by Tosoh Corporation was partially improved and used. The gradient changed from eluent A 100% to eluent B 100% in 30 minutes. The total flow rate of the pumps (2, 16) is set to 200 μL per minute, and the flow rate value of the B side channel is made constant at 176.5 μL per minute with the thermal flow meter (5a) and the flow control device (5b). Control was performed so that 23.5 μL per minute, which is the above difference, flows through the A-side flow channel for analysis.

  As measurement samples, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, n-butyl p-hydroxybenzoate, hexyl p-hydroxybenzoate, heptyl p-hydroxybenzoate 6 A mixture of seeds was used.

(B) Micro flow rate liquid feeding method of the present invention FIG. 17 shows a system configuration of a micro flow rate liquid feeding device by the active split method of the present invention. The eluent A (1) is sent by the liquid feed pump A (2), and the eluent B (15) is sent by the liquid feed pump B (16). Solvent gradient is performed by changing the flow rates of the liquid feed pump A (2) and the liquid feed pump B (16) (the sum of the flow rates of the liquid feed pump A (2) and the liquid feed pump B (16) is constant). The sent eluent (1, 15) passes through the first thermal flow meter (4) and is then branched into the A-side flow path and the B-side flow path by the branch block (13). The A-side channel is a channel used for analysis, and a sample injection device (6), an analysis column (7), a column thermostat (8), and a detector (9) are arranged. A second thermal type flow meter (5a) and a flow rate control device (5b) are arranged in the B side flow path. The A-side channel is a channel through which a minute flow rate of several tens of μL per minute to several hundreds of nL per minute flows. The B-side channel is a channel through which most of the rest flows.

  The measurement conditions were an analytical column (7), an ODS column (manufactured by Tosoh Corporation) having an inner diameter of 1 mm, a length of 50 mm and a particle size of 3 μm, and an eluent (1, 15) as A: acetonitrile / water (20/80), B : Acetonitrile / water (70/30) was used. As a pump (2, 16), CCSO manufactured by Tosoh Corporation was partially improved and used. The gradient changed from eluent A 100% to eluent B 100% in 30 minutes. For the eluent, the flow rate value is measured by the first thermal flow meter (4), and the value (flow rate value) multiplied by the flow rate ratio (0.883) is calculated by the calculator (14). And the control signal which makes the said calculated value and the flow value of the 2nd thermal type flow meter (5a) correspond from the said calculator (14) is sent to the flow control apparatus (5b), and the flow volume of B side flow path The above difference is applied to the A-side flow path for analysis (the flow rate of the first thermal flow meter) so that the value becomes μL per minute (flow value of the first thermal flow meter × 0.883) μL. Value × 0.117) μL was controlled.

  As the measurement sample, the same as in the case where the system configuration (A) (FIG. 16) was used, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and n-butyl p-hydroxybenzoate. , Hexyl p-hydroxybenzoate and heptyl p-hydroxybenzoate were mixed.

(C) Results FIG. 18 shows chromatogram results when both system configurations (A) and (B) are used (FIGS. 16 and 17). In FIG. 18, a is the result when the system configuration (A) (FIG. 16) is used, and b is the result when the system configuration (B) (FIG. 17) is used. When the system configuration (B) (FIG. 17) is used, all six types of peaks can be confirmed, indicating that the gradient is performed well (FIG. 18b). On the other hand, when the system configuration (A) (FIG. 16) was used, elution was extremely delayed, and only three types of peaks could be confirmed even at 80 minutes (FIG. 18a). From the result of FIG. 18a, it can be seen that when the system configuration (A) (FIG. 16) is used, the flow rate is extremely reduced from the middle of the analysis.

  The column pressure change at this time is shown in FIG. 19, the output value of the first thermal flow meter (4) (flow rate before branching) is shown in FIG. 20, and the output value of the second thermal flow meter (5a) (B The flow rate of the side flow path is shown in FIG. 21 (each using a system configuration (FIG. 16) in which (a) in the figure is a system configuration (FIG. 17) in which (b) is (B). Is the result of when).

  (A) (B) When both system configurations (FIGS. 16 and 17) are used, the output value (flow rate before branching) of the first thermal flow meter (4) is linear as the gradient progresses. It decreases (FIG. 20). This is because the actual flow rate does not change, but the specific heat of the solvent changes, so the response of the thermal flow meter changes.

  When the system configuration (A) (FIG. 16) is used, the gradient starts because the control is performed so that the output value of the second thermal flow meter (5a) (the flow rate of the B side channel) is constant. After a while, it becomes a constant value, but after 13 minutes, a decrease in the value is seen, indicating that it is not controlled correctly (FIG. 21a).

  On the other hand, when the system configuration (B) (FIG. 17) is used, the actual flow value is measured with the first thermal flow meter (4), and the value (actual flow value) is multiplied by the branching ratio. Since control is performed to match the flow rate value with the flow rate value of the second thermal flow meter (5a), the output flow rate value of the second thermal flow meter (5a) (the flow rate of the B side channel) Decreases in synchronization with a decrease in the output flow rate value (flow rate before branching) of the first thermal flow meter (4) (FIG. 21b).

  FIG. 22 shows the difference between the output flow value of the first thermal flow meter (4) and the output flow value of the second thermal flow meter (5a), that is, the flow rate of the A-side flow path used for analysis. a is the flow rate when using the system configuration (A) (FIG. 16), b is the flow rate when using the system configuration (B) (FIG. 17), and c is the change in specific heat (theoretical value). Show. The specific heat of c has a delay of 2 minutes in consideration of the influence of voids.

  When the system configuration of FIG. 16A (FIG. 16) is used (FIG. 22a), it can be seen that after the start of the gradient, it rapidly decreases from about 2 minutes and does not flow at all after about 13 minutes. On the other hand, when the system configuration (B) (FIG. 17) is used (FIG. 22b), at the start of the gradient, what was about 27 μL per minute gradually decreases, and after 30 minutes, it becomes about 17 μL per minute. It has become.

  Even when the system configuration (B) (FIG. 17) is used (FIG. 22b), the calculated flow rate is reduced because the response of the thermal flow meter is changed due to the change in specific heat. The actual flow rate can be calculated by dividing the difference between the output flow rate value of the first thermal flow meter (4) of FIG. 20 and the output flow rate value of the second thermal flow meter (5a) by the specific heat. FIG. 23 shows the flow rate of the A-side channel after division. As described above, when the minute flow rate liquid feeding method of the present invention is used, it is understood that a substantially constant flow rate is obtained at about 30 μL per minute during the gradient execution.

  The reproducibility when the system configuration (B) (FIG. 17) is used is shown in FIGS. 24 shows the chromatogram (n = 10), FIG. 25 shows the change in elution time (n = 10), and FIG. 26 shows the reproducibility of the elution time. (N = 10 × 2). Thus, the reproducibility of the elution time of each peak is as good as 0.1 to 0.5% in Cv (%) (FIG. 26). By using the flow rate control method of the present invention, A It is suggested that a minute flow rate can be accurately fed to the side channel.

Example 3 Capillary Electrophoresis Device Using the Microflow Rate Liquid Feeding Method of the Present Invention In Examples 1 and 2, an example in which the present invention is applied to liquid chromatography in which a liquid is fed by a pump is shown. The same liquid feeding method can be applied to this. FIG. 27 shows the configuration. An electrolytic solution A (20) is arranged in place of the liquid feed pump (2), and a conduit is inserted. The waste liquid of the A side channel and the waste pipe of the B side channel are inserted into the electrolyte B (21). By inserting the electrodes (22) and (23) into the electrolytic solution A (20) and the electrolytic solution B (21), respectively, and connecting to the power source (18), a liquid flow is generated, and capillary electrophoresis can be performed. .

Flow path diagram (isocratic method) when a small flow rate is sent by the basic split method (static split method). A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. Flow diagram when a minute flow rate is sent by the conventional active split method (isocratic method). A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. The graph which showed flow control in the case of the eluent composition not changing in the conventional active split method (isocratic elution method). The horizontal axis represents time, and the vertical axis represents the flow rate. (A) is a graph showing changes in the flow rate before branching and the B-side flow path, the solid line is the flow rate before branching, and the solid area is the control value of the thermal flow meter / flow control device for the B-side flow path. Show. (B) is the graph which showed the change of the flow volume of the A side flow path, and the filled area shows the flow volume of the A side flow path. Flow path diagram when the second thermal flow meter (5a) and the flow control device (5b) are arranged in front of the sample injection device (6) on the A side flow path in the micro flow rate liquid feeding method of the present invention. . A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. The flow-path figure at the time of arrange | positioning the 2nd thermal type flow meter (5a) and the flow control apparatus (5b) after the detector (9) of an A side flow path in the micro flow volume liquid feeding method of this invention. A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. FIG. 5 is a flow chart (isocratic method) when the second thermal flow meter (5a) and the flow control device (5b) are arranged in the B-side flow path in the minute flow rate liquid feeding method of the present invention. A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. The graph which showed flow control in the case of the eluent composition not changing in the micro flow volume liquid feeding method of this invention (isocratic elution method). The horizontal axis represents time, and the vertical axis represents the flow rate. (A) is a graph showing changes in the flow rate before branching and the B-side flow path, the solid line is the flow rate before branching, and the solid area is the control value of the thermal flow meter / flow control device of the B-side flow path. Indicates. (B) is the graph which showed the change of the flow volume of the A side flow path, and the filled area shows the flow volume of the A side flow path. Changes in the output of the thermal flow meter when using a thermal flow meter corrected with water and performing a gradient from 100% water to 100% acetonitrile. The horizontal axis represents the concentration of acetonitrile, the left vertical axis represents the flow rate, and the right vertical axis represents the specific heat. The broken line indicates the actual flow rate, and the filled area indicates the flow value indicated by the thermal flow meter. The graph which showed flow control by the conventional active split method when an eluent composition changes (gradient elution method). The horizontal axis represents time, and the vertical axis represents the flow rate. (A) is a graph showing changes in the flow rate before branching and on the B-side channel, the solid line is the flow rate before branching indicated by the thermal flow meter, the broken line is the actual flow rate, and the painted area is the B-side channel. The control value of the thermal type flow meter / flow rate control device is shown. (B) is the graph which showed the change of the flow volume of the A side flow path, and the filled area shows the flow volume of the A side flow path. The graph which showed flow volume control in case the eluent composition changes by the micro flow volume liquid feeding method of this invention (gradient elution method). The horizontal axis represents time, and the vertical axis represents the flow rate. (A) is a graph showing changes in the flow rate before branching and on the B-side channel, the solid line is the flow rate before branching indicated by the thermal flow meter, the broken line is the actual flow rate, and the painted area is the B-side channel. The control value of the thermal type flow meter / flow rate control device is shown. (B) is the graph which showed the change of the flow volume of the A side flow path, and the filled area shows the flow volume of the A side flow path. Change in the output value of the thermal flow meter on the B side channel when a minute flow rate is sent by the conventional active split method. The horizontal axis represents time, and the vertical axis represents the flow rate. Chromatogram result (n = 20) when a minute flow rate is fed by the conventional active split method. The horizontal axis represents time, and the vertical axis represents absorbance. The flow rate change of each flow path when a minute flow rate is fed by the minute flow rate feeding method of the present invention. The horizontal axis represents time, and the vertical axis represents the flow rate. (A) shows the change of the output value of the 1st thermal type flow meter arrange | positioned at the flow path before a branch. (B) shows the change of the output value of the 2nd thermal type flow meter arrange | positioned at the B side flow path. (C) shows the difference of the output value of the 2nd thermal type flow meter arrange | positioned at the B side flow path from the output value of the 1st thermal type flow meter arrange | positioned at the flow path before a branch. The chromatogram (n = 20) when feeding a micro flow rate by the micro flow rate liquid feeding method of this invention. The horizontal axis represents time, and the vertical axis represents absorbance. The result which showed Cv (%) of elution time (3 batch measurement at n = 10). (A) shows Cv (%) when a minute flow rate is fed by the conventional active split method. (B) shows Cv (%) when a minute flow rate is fed by the minute flow rate feeding method of the present invention. Flow diagram (liquid gradient elution method) when a minute flow rate is sent by the conventional active split method. A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. FIG. 4 is a flow chart (solvent gradient elution method) when a minute flow rate is fed by the minute flow rate feeding method of the present invention. A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path. In the solvent gradient elution method, a chromatogram comparing when a minute flow rate is fed by the conventional active split method and when a minute flow rate is fed by the minute flow rate feeding method of the present invention. The horizontal axis represents time, and the vertical axis represents absorbance. a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. In the solvent gradient elution method, the figure which showed the change of column pressure when feeding a micro flow rate by the conventional active split method, and when sending a micro flow rate by the micro flow rate feeding method of this invention. The horizontal axis represents time, and the vertical axis represents column pressure. a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. In the solvent gradient elution method, when a minute flow rate is fed by the conventional active split method, and when a minute flow rate is fed by the minute flow rate feeding method of the present invention, it is arranged in the channel before branching. The figure which showed the change of the output value of a 1st thermal type flow meter. The horizontal axis represents time, and the vertical axis represents the flow rate (the output value of the flow meter). a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. In the solvent gradient elution method, the second flow rate disposed in the B-side flow path when the minute flow rate is fed by the conventional active split method and when the minute flow rate is fed by the minute flow rate feeding method of the present invention. The figure which showed the change of the output value of the thermal type flow meter. The horizontal axis represents time, and the vertical axis represents the flow rate. a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. In the solvent gradient elution method, when the minute flow rate is fed by the conventional active split method, and when the minute flow rate is fed by the minute flow rate feeding method of the present invention, the first flow channel arranged before branching is arranged. The figure which showed the difference of the output value of the 2nd thermal type flow meter arrange | positioned in the B side flow path from the output value of one thermal type flow meter. The horizontal axis represents time, and the vertical axis represents the flow rate. a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. c shows the change in the specific heat of the eluent (with a delay of 2 minutes considering the effect of voids). In the solvent gradient elution method, the difference in the output value of the thermal flow meter between when the minute flow rate is fed by the conventional active split method and when the minute flow rate is fed by the minute flow rate feeding method of the present invention. The figure which showed the value divided by the specific heat. The horizontal axis represents time, and the vertical axis represents the flow rate. a shows the result when a minute flow rate is fed by the conventional active split method. b shows the result when a minute flow rate is fed by the minute flow rate feeding method of the present invention. The chromatogram result (n = 10) when feeding a minute flow rate by the minute flow amount feeding method of the present invention. The horizontal axis represents time, and the vertical axis represents absorbance. The figure which showed the fluctuation | variation of the elution time when a minute flow rate is sent with the minute flow amount feeding method of the present invention (n = 10). The figure which showed Cv (%) of elution time when supplying a micro flow rate by the micro flow rate liquid feeding method of this invention (2 batch measurement at n = 10). FIG. 3 is a flow chart when the micro flow rate liquid feeding method of the present invention is applied to capillary electrophoresis. A long chain line indicates the A-side flow path, and a long broken line indicates the B-side flow path.

Explanation of symbols

1. Eluent A
2. Liquid feed pump A
3. 3. Pressure gauge First thermal flow meter 5a. Second thermal flow meter 5b. 5. Flow control device 6. Sample injection device Analytical column 8. Column thermostat 9. Detector 10. Waste liquid A
11. Waste B
12 Feedback signal 13. Branch block 14. Calculator 15. Eluent B
16. Liquid feed pump B
17. Variable resistor 18. Power supply 19. Electrophoretic separation medium 20. Electrolyte A
21. Electrolyte B
22. Electrode A
23. Electrode B

Claims (6)

A micro flow rate liquid feeding device for flowing a liquid at a small flow rate to a separation medium by flowing a liquid by a liquid feeding unit and branching it to a separation medium side channel and a waste side channel by using a branching unit. ,
A first flow meter for measuring a flow rate value of the liquid flowing between the liquid feeding means and the branching means at an arbitrary sampling interval;
A second flow meter that measures the flow rate value of the liquid flowing in the separation medium side flow path or the waste side flow path at the same or different sampling interval as the first flow meter;
A flow rate control means for controlling the flow rate of the liquid flowing in the flow path on the separation medium side or the flow path on the waste side as necessary;
Based on the flow rate values measured by the first and second flow meters, the flow rate of the liquid flowing in the flow path between the liquid feeding means and the branching means, the flow path on the separation medium side, and the flow path on the disposal side is calculated. Arithmetic means for sending a signal for control,
The calculation means (1-1) uses the flow value measured by the first flow meter as an input value, multiplies the flow value by an arbitrary value less than 1, and the flow rate to be measured by the second flow meter. Calculate the value
(1-2) Using the flow value measured by the second flow meter as an input value, comparing it with the flow value to be measured by the calculated second flow meter, and making both flow values coincide, Controlling the flow rate control means;
Or
(2-1) Using the flow value measured by the second flow meter as an input value, multiply the flow value by an arbitrary value exceeding 1, and calculate the flow value to be measured by the first flow meter. ,
(2-2) Using the flow value measured by the first flow meter as an input value, comparing the flow value to be measured by the first flow meter calculated above, and matching both flow values, Controlling the liquid feeding means;
The micro flow rate liquid feeding device characterized by the above. The minute flow rate liquid feeding device according to claim 1, wherein the first and second flow meters are thermal flow meters. The minute flow rate liquid feeding device according to claim 1, wherein the second flow meter and the flow rate control means are installed in a flow path on the disposal side. In the calculation means, in the time measured at each sampling interval,
(1) Flow rate values measured by the first and second flow meters,
(2) a calculated value of the flow rate to be measured by the first or second flow meter,
(3) Flow rate value to be controlled by the flow rate control means or the liquid feeding means,
(4) Information relating to control of the flow rate control means or the liquid feeding means,
4. The micro flow rate liquid feeding device according to claim 1, further comprising recording means for recording This is a micro flow rate feeding method in which a liquid is flowed using a liquid feeding means and branched to a flow path on the separation medium side and a waste side flow path using a branching means, thereby allowing a liquid with a small flow rate to flow into the separation medium. And
Measure the flow rate value of the liquid flowing between the liquid feeding means and the branching means and the flow rate value of the liquid flowing in the separation medium side flow path or the disposal side flow path,
(1-1) The flow rate value of the liquid flowing between the liquid feeding means and the branching means is multiplied by an arbitrary value less than 1, and the liquid to be flowed to the separation medium side flow path or the waste side flow path Calculate the flow value,
(1-2) The calculated flow rate value of the liquid that should flow through the separation medium side channel or the disposal side channel and the flow rate value of the liquid flowing through the separation medium side channel or the disposal side channel. Control the flow rate of the liquid flowing in the separation medium side flow path or the waste side flow path so as to match.
Or (2-1) multiplying the flow rate of the liquid flowing in the flow path on the separation medium side or the flow path on the waste side by an arbitrary value greater than 1, and the liquid to flow between the liquid feeding means and the branching means Calculate the flow value,
(2-2) The flow rate value of the liquid that should flow between the liquid feeding unit and the branching unit and the flow rate value of the liquid flowing between the liquid feeding unit and the branching unit are matched. Controlling the flow rate of the liquid flowing between the liquid means and the branching means,
The method for feeding a minute flow rate as described above. The flow rate controlled by a calculated value obtained by multiplying the flow rate value of the liquid flowing between the liquid feeding unit and the branching unit by an arbitrary value less than 1 is the flow rate of the liquid flowing in the waste-side flow path. The micro flow rate liquid feeding method according to claim 5.

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