JP2013185897A - Minute flow rate liquid feeding method and device utilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a minute flow rate liquid feeding method capable of suppressing flow fluctuation for a long time and flow fluctuation for a short time and providing a stable liquid feeding state in a short period of time, and having a high responsibility, and a liquid feeding device having a simple configuration utilizing the method.SOLUTION: In a liquid feeding method for measuring the flow rate of a liquid fed by liquid feeding means and performing liquid feeding control by the liquid feeding means on the basis of the measured flow rate, the liquid feeding control by the liquid feeding means instructs the liquid feeding means to feed a flow rate higher than that to be fed by the liquid feeding means. When the measured flow rate becomes a flow rate higher than the flow rate of the liquid to be fed by a fixed value, the liquid feeding control instructs the liquid feeding means to stop liquid feeding. When the measured flow rate becomes a flow rate lower than the flow rate of the liquid to be fed by a fixed value, the liquid feeding control instructs the liquid feeding means to resume liquid feeding. The problem is solved by this liquid feeding method and a liquid feeding device utilizing the method.

Description

  The present invention relates to a method capable of stably feeding a minute flow rate of several μL to several tens of μL per minute, and an apparatus using the method.

  In the field of liquid chromatography, so-called “micronization”, in which the inner diameter of the column is reduced, is progressing from the viewpoint of reducing the eluent. In general liquid chromatography, a column having an inner diameter of about 4.6 mm is used, and analysis is performed at a flow rate of about 1 mL per minute. On the other hand, in “micro-sized” liquid chromatography, a column having an inner diameter of several hundred μm to several mm is used, and analysis is performed at a flow rate of several μL to several tens μL per minute.

  FIG. 1 shows a configuration diagram of a general liquid chromatograph. The liquid chromatograph detects a pump (1) for feeding an eluent (10), a sample introduction valve (4) for introducing a sample (6), an analysis column (7) for separating the sample, and a separated component. A detector (9) is provided. When “micronizing” the liquid chromatograph shown in FIG. 1, not only miniaturization and performance improvement of the analytical column (7), but also other mechanisms are required to be miniaturization and performance improvement. Among them, the liquid feeding means including the liquid feeding pump (1) is an important element. The liquid feeding means provided in a general liquid chromatograph need only be able to send liquid stably at a flow rate of about 1 mL per minute, whereas the liquid feeding means provided in a “micro-sized” liquid chromatograph requires several minutes per minute. This is because it is necessary to stably feed liquid at a flow rate of μL to several tens of μL.

  As a liquid feed pump provided in a liquid chromatograph, a check valve (21) that normally suppresses the flow of fluid in one direction is arranged above and below the pump chamber, and fluid is sucked / discharged by reciprocating movement of the plunger (20). Again, use a plunger pump (Figure 2). The plunger (20) is reciprocated by converting the rotational motion of a cam (22) attached to the motor shaft directly or via a pulley belt drive (FIG. 3). When an eccentric cam with a simple structure is used as the cam (22) and the motor (25) is rotated at a constant speed, the plunger (20) reciprocates in a form in which the displacement-time curve draws a sine curve. The liquid is sucked / discharged in synchronism (reciprocating type: FIGS. 3a and 4a). In the reciprocating method, the time required for sucking the liquid and the time required for discharging the liquid are the same, and the influence of the pulsation appears greatly in the chromatogram. In order to solve this problem, as the cam (22), a linear cam is used for which the discharge region has a linearity over a long time while the suction region returns in a short time, and the cam is rotated at a constant speed. Thus, there is a method of reciprocating the plunger (20) (quick return type: FIGS. 3b and 4b). In the quick return method, the time required for discharging the liquid is long, while the time required for sucking the liquid is short, so that the influence of pulsation in the chromatogram can be suppressed as compared with the reciprocating method.

  In the plunger pump, the amount of liquid feeding can be increased (high flow rate) by increasing the number of rotations of the motor that rotates the cam, and the amount of liquid feeding can be decreased (low flow rate) by decreasing the number of rotations. FIG. 5 (reciprocal type: FIG. 5a, quick return type: FIG. 5b) shows the plunger operation and pressure fluctuation of the plunger pump at a high flow rate, and FIG. 6 (reciprocation) shows the plunger operation and pressure fluctuation of the plunger pump at a low flow rate. Formula: FIG. 6a, quick return formula: FIG. 6b), respectively. Regardless of the reciprocating type or quick return type, the cam shape is fixed and the motor rotation speed is constant. Therefore, if the motor rotation speed is reduced (that is, the flow rate is reduced), liquid is sucked. The time becomes longer and the time for the pressure to drop also becomes longer. Therefore, the flow rate stability cannot be ensured in the low flow rate region, and it is difficult to obtain good analysis accuracy.

  In order to ensure flow rate stability even when a low flow rate is supplied by a plunger pump, attempts have been made to make the rotation speed of the motor that rotates the cam variable. That is, the cam rotation speed at the time of liquid suction is made higher than the rotation speed of the cam at the time of liquid discharge, thereby shortening the pressure drop time and suppressing the influence of pulsation in the chromatogram (FIG. 7). reference). Moreover, in order to perform the said method more simply, the pump which reciprocates a plunger using a ball screw (23) instead of a cam is disclosed (nonpatent literature 1). Since the pump can discharge the liquid by rotating the ball screw (23) forward and can suck the liquid by reversing the ball screw (23), the liquid suction / discharge control is easy. (See FIGS. 8 and 9). However, the pump is liable to be troubled by a small amount of foreign matters and bubbles contained in the eluent, and sufficient care must be taken when using it.

  As another method for securing the flow rate stability at the time of low flow rate liquid feeding, there is a method using a syringe pump (13) (FIG. 10). It has a cylinder and a piston with a cylinder volume that is larger than the volume of the eluent required for one analysis, and is connected to the analysis system after filling the cylinder with the eluent while disconnected from the analysis system. This is a method of discharging slowly. By using a syringe pump, liquid feeding in a low flow rate region can be performed very stably. However, there is a problem in that the time required for stabilization becomes long because the cylinder volume with respect to the liquid supply amount increases when the liquid supply amount becomes very small (for example, several μL to several tens μL per minute). Another problem is that syringe pumps are not suitable for continuous measurement.

  As yet another method for ensuring the flow rate stability at the time of low flow rate feeding, as a plunger pump, a splitter used in a general liquid chromatograph (for example, a flow rate of several hundred μL per minute) is used. There is a method of creating a minute flow rate (FIG. 11). That is, after the liquid is fed at a flow rate of several hundreds μl per minute by the liquid feed pump (1), the amount of 1/10 to 1/100 is led to the analysis column (7) by the splitter. Examples of the splitter include those using a simple resistance ratio using a resistance tube (11) shown in FIG. 11, and those using a controlled valve mechanism. However, there are many problems such as lack of stability and disposal of most of the eluent.

  In order to solve these problems, a method of controlling the liquid feed amount (flow rate) by the liquid feed pump to be constant by feeding back the value of the liquid feed amount by the liquid feed pump to the control unit of the liquid feed pump (FIGS. 12 to 14). Specifically, a flow meter (15) capable of measuring the amount of liquid fed by the liquid feed pump (1) is provided on the discharge side of the liquid feed pump (1), and the flow meter (15) using the controller (3). The liquid feed amount of the liquid feed pump is finely adjusted by performing proportional control by PID control or the like so that the value of becomes the specified value. By this method, it becomes possible to stably feed a minute flow rate. However, there is a problem that it takes a long time until the flow rate becomes constant, or the waviness remains after stabilization as shown in FIG. Further, since the flow rate is finely adjusted by proportional control, there is a drawback that it is difficult to follow the flow rate fluctuation in a short time, although the correction force for the flow rate fluctuation in the long term is strong.

In the case of liquid feeding at a minute flow rate, it is a big problem that a huge amount of time is required from the start of liquid feeding until the pressure becomes constant. This is because, when the liquid feeding amount is low, the ratio of the void volume of the liquid feeding pump or the like increases with respect to the liquid feeding amount, so that the pressure does not increase easily even when the liquid feeding is started. To solve the problem, increase the pressure in a short time by sending it at a flow rate higher than the flow rate actually used until the pressure rises, and then return it to the flow rate used in the analysis. A method is mentioned (refer FIG. 15). However, when the above method is adopted, it is necessary to set in advance a pressure value necessary for starting the analysis. If this setting is mistaken, there is a possibility that the analysis column is seriously damaged.
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Uniflows Co., Ltd., homepage of embedded micropump for liquid delivery http://www.uniflows.co.jp/

  As described above, the method of stably feeding a minute flow rate includes a method of mechanically stabilizing the minute flow rate, and a method of feeding back the flow rate value to the solution feeding means and keeping the amount of fluid fed constant by proportional control. And divided. The former has problems such as structural complexity, and the latter has problems such as poor response to short-term fluctuations, so that the analyst cannot easily use it.

  Therefore, the present invention suppresses flow rate fluctuations in a long period and flow rates in a short period, and a stable liquid delivery state can be obtained in a short time, and a highly responsive micro flow rate liquid delivery method, and a configuration using the method An object of the present invention is to provide a simple liquid delivery device.

  The present invention made in view of the above problems includes the following inventions.

The first aspect of the present invention is:
A liquid feeding method for measuring the flow rate of the liquid fed by the liquid feeding means and performing liquid feeding control by the liquid feeding means based on the measured flow rate,
The liquid feeding control by the liquid feeding means instructs the liquid feeding means to feed a flow rate higher than the flow rate of the liquid to be fed by the liquid feeding means, and the measured flow rate should be the liquid feeding. When the flow rate is higher than the liquid flow rate by a certain value or more, the flow rate is instructed to stop by the liquid feeding means, and the measured flow rate is lower than the liquid flow rate to be fed by a certain value or more. In this case, the liquid feeding method is a control for instructing to resume the liquid feeding by the liquid feeding means.

The second aspect of the present invention is:
Based on the liquid feeding means for feeding the liquid, the flow rate measuring means for measuring the flow rate of the liquid fed by the liquid feeding means provided on the discharge side of the liquid feeding means, and the flow rate measured by the flow rate measuring means A liquid feeding device provided with a control means for performing liquid feeding control in the liquid feeding means,
A flow rate determination means for determining whether the flow rate measured by the flow rate measurement means is a flow rate greater than a certain value or less than a flow rate of the liquid to be delivered by the liquid delivery means;
The control means instructs the liquid feeding means to send a flow rate higher than the flow rate of the liquid to be fed by the liquid feeding means, and the flow rate measured by the flow rate measuring means is the flow rate of the liquid to be delivered. When the flow rate determination means determines that the flow rate is more than a certain value, the flow rate is measured by the flow rate measurement means, and the flow rate measured by the flow rate measurement means is greater than the flow rate of the liquid to be sent. If the flow rate determining means determines that the value is less than a certain value, the liquid feeding device is a means for instructing the liquid feeding means to resume liquid feeding.

  A third aspect of the present invention is the liquid delivery device according to the second aspect, wherein the flow rate measuring means is a thermal flow meter.

  According to a fourth aspect of the present invention, there is provided a liquid feeding part for feeding an eluent, a sample introduction part for introducing a sample, an analysis column part for separating each component in the introduced sample, and elution from the analysis column part A liquid chromatograph including a detection unit that detects each of the components, wherein the liquid feeding unit is the liquid feeding device according to the second or third aspect.

  Hereinafter, the present invention will be described in detail.

A flow chart showing the liquid feeding method of the present invention is shown in FIG. In the liquid feeding method of the present invention,
A target flow rate F (T) which is a flow rate of the liquid to be fed by the liquid feeding means,
Drive flow rate: F (S), which is a set value of the flow rate that is actually delivered by the solution delivery means,
A control upper limit flow rate F (H) which is a flow rate (threshold value) for stopping liquid feeding by the liquid feeding means,
Control lower limit flow rate F (L) which is a flow rate (threshold value) for resuming liquid feeding by the liquid feeding means,
These four parameters are set and a minute flow rate is sent.

  In the liquid feeding method of the present invention, the driving flow rate F (S) is set to a value larger than the target flow rate F (T). As a method of setting the driving flow rate F (S), a constant value may be added to the target flow rate F (T) or may be set by multiplying a constant value. Further, the addition value or the multiplication value may be changed according to the flow rate, or may be set in combination with addition and multiplication. Further, in a high flow rate region where the influence of pulsation or the like is small (for example, several hundred μL or more per minute), it may be set not to add to the target flow rate F (T). An example of the relationship between the target flow rate F (T) and the drive flow rate F (S) in the liquid feeding method of the present invention is shown in FIGS.

  FIG. 17a is a diagram showing an example in which the drive flow rate F (S) is set by adding a certain value to the target flow rate F (T) over the entire range of the target flow rate F (T).

F (S) = F (T) + k1 * k1: Additional flow rate Figure 17b shows the target flow rate F (T) divided into two sections with a constant flow rate (m1) as the boundary. The driving flow rate F (S) is set so as to be equal to or higher than the flow rate F (T), and in a high flow rate region where the flow rate is equal to or higher than m1, the constant value is added to the target flow rate F (T). ) Is a diagram illustrating an example of setting.

0 ≦ F (T) <m1: F (S) = k2
F (T) ≧ m1: F (S) = F (T) + k3
* K2: Drive flow rate in the low flow rate range, k3: Addition flow rate in the high flow rate range
m1: Section flow point of the target flow rate FIG. 18c divides the target flow rate F (T) into two sections with a constant flow rate (m2) as a boundary, and in a low flow rate region less than the flow rate m2, the target flow rate F depends on the flow rate. A value obtained by adding (T) based on the following formula is set as the driving flow rate F (S), and the addition to the target flow rate F (T) is not performed in a high flow rate range of the flow rate m2 or more (that is, the target flow rate F (T)). And the drive flow rate F (S) coincides with each other).

0 ≦ F (T) <m2: F (S) = {(m2−k4) / m2} × F (T) + k4
F (T) ≧ m2: F (S) = F (T)
* K4: Additional flow rate in the low flow rate range, m2: Section point of the target flow rate The control upper limit flow rate F (H) is set by adding a certain value to the target flow rate F (T), and the control lower limit flow rate F (L ) May be set by subtracting a certain value from the target flow rate F (T). When the addition value and the subtraction value to the target flow rate F (T) are set small, Flow (liquid feeding) / Stop (stop) by the liquid feeding means (pump) is frequently repeated, so that the stability and accuracy of the liquid feeding are improved. It is preferable in terms of increase. In practice, however, there is a limit to reducing the added value and the subtracted value to the target flow rate F (T) because of the resolution of the pump flow rate control. Therefore, optimal addition values and subtraction values may be set through preliminary tests.

  In the liquid feeding method of the present invention, the liquid is first fed by the liquid feeding means at the driving flow rate F (S) set by the above-described method. When the flow rate of the fed liquid is measured and the value F (R) exceeds the control upper limit flow rate F (H), the liquid feeding by the liquid feeding means is stopped. When the liquid feeding by the liquid feeding means is stopped, the flow rate value F (R) decreases. When the flow rate value F (R) falls below the control lower limit flow rate F (L), liquid feeding by the liquid feeding means is resumed. By repeating this operation, stable liquid feeding near the target flow rate F (T) is performed (see FIG. 19). In the liquid feeding method of the present invention, the driving flow rate F (S) is set to a value larger than the target flow rate F (L). Therefore, when the flow rate value F (R) falls below the control lower limit flow rate F (L), when the liquid feeding by the liquid feeding means resumes, the flow rate value F (R) quickly reaches the control upper limit flow rate F (H), When liquid feeding by the liquid feeding unit stops, the flow rate value F (R) quickly reaches the control lower limit flow rate F (L), and liquid feeding by the liquid feeding unit is resumed. As a result, Flow (stopping) / Stop (stopping) by the liquid feeding means is repeated frequently (for example, several times per second), and extremely stable liquid feeding near the target flow rate F (T) is realized. Is done.

  The liquid feeding method of the present invention also has an effect of greatly shortening the time until the pressure becomes constant. FIG. 20 schematically shows the effect. In a conventional liquid feeding method that does not perform feedback control, liquid feeding by the liquid feeding means is always performed at a target flow rate value. Therefore, in the conventional method, when a minute flow rate is to be fed, the time for sucking the liquid by the liquid feeding means also becomes longer at the same time, and a large pressure fluctuation occurs until the pressure becomes constant (until the target flow rate is reached). On the other hand, in the liquid feeding method of the present invention, the liquid is fed at a flow rate (driving flow rate) larger than the target flow rate from the start of liquid feeding. Therefore, the liquid is fed at a higher speed than the conventional method, and as a result, the time until the pressure becomes constant (until the target flow rate is reached) can be greatly shortened.

  FIG. 21 shows an example of a liquid feeding apparatus (hereinafter simply referred to as the liquid feeding apparatus of the present invention) using the liquid feeding method of the present invention. The apparatus of FIG. 21 includes a liquid feed pump (1) for feeding an eluent (10), a flow meter (15) for measuring the flow rate of the eluent, a flow rate determining means (16), and a liquid feed pump (1 And a pump controller (3) for controlling liquid suction / discharge.

  As the liquid feeding pump (1) provided in the liquid feeding device of the present invention, a general reciprocating plunger pump or a quick return plunger pump (FIGS. 2 and 3) which is driven by rotating a cam at a constant speed may be used. However, using a plunger pump (FIG. 7) in which the rotation speed of the motor for rotating the cam is variable and the cam rotation speed during liquid suction is higher than the rotation speed of the cam during liquid discharge, or a ball screw is used instead of the cam. By using a plunger pump that reciprocates the plunger (Figs. 8 and 9), the suction operation can be performed at a high speed even when a minute flow rate is supplied, and fluctuations in the flow rate and the associated pressure fluctuations can be minimized. It is preferable at the point which can deliver liquid. FIG. 22 shows a schematic diagram when the liquid feeding method of the present invention is performed using the plunger pump shown in FIG.

  The flow meter (15) provided in the liquid feeding device of the present invention is not particularly limited as long as it is a flow meter capable of measuring a minute flow rate (specifically, about several μL per minute), but a Coriolis type flow meter or a thermal type. A flow meter that can directly measure a mass flow rate (g / min) such as a flow meter is preferable in that a minute flow rate can be measured with high accuracy. Among them, the thermal flow meter is particularly preferable as the flow meter provided in the liquid feeding device of the present invention in that the internal capacity can be made extremely small, the structure is relatively simple, and the pressure resistance is relatively easy to secure. . FIG. 34 shows the principle of the thermal flow meter. A heater (29) for supplying a constant amount of heat is wound around the pipe (30) through which the liquid flows, and when the liquid is not flowing, the position of T1 and the position of T2 are adjusted to the same temperature. When the liquid flows, the temperature at the position of T1 decreases because the liquid loses heat, while the temperature at the position of T2 increases by the heater (29). Since the temperature difference between the T1 position and the T2 position is proportional to the liquid mass flow rate, the temperature detection unit (28) detects the temperature at the T1 position and the T2 position to increase the liquid mass flow rate. It can be measured with high accuracy. Thermal flow meters are susceptible to environmental temperature fluctuations because they are affected by the amount of heat of the fluid. For this reason, when the thermal flow meter is provided in the liquid feeding device of the present invention, it is preferable that at least the temperature detection unit (28) and the heater (29) are provided in a thermostat and the ambient temperature is kept constant. In the thermal type flow meter, the measured value of the volume flow rate also changes when the density and specific heat of the liquid to be measured are different. For example, the specific heat of acetonitrile usually used as an eluent for liquid chromatography is 1.27 J / ° C. · g, which is very different from the specific heat of water (4.19 J / ° C. · g). Therefore, as a result of feeding 1 mL / min of water, when 1 mL / min of acetonitrile is fed with a thermal flow meter showing a measured value of 1 mL / min, the measured value becomes 0.304 mL / min. Therefore, when the thermal flow meter is provided in the liquid delivery device of the present invention, it is necessary to correct the flow value in advance with the liquid to be used. After the flow rate measured by the flow meter (15) is compared / computed with the flow rate set by the flow rate determination means (16), a control signal is sent to the pump controller (3), and the liquid is sent based on the control signal. The suction / discharge operation of the pump (1) is performed.

  The liquid feeding method of the present invention is a liquid feeding method for measuring the flow rate of the liquid fed by the liquid feeding means and performing liquid feeding control by the liquid feeding means based on the measured flow rate, wherein the liquid feeding method The liquid feeding control by the means instructs the liquid feeding means to send a flow rate higher than the flow rate of the liquid to be fed by the liquid feeding unit, and the measured flow rate is the flow rate of the liquid to be fed. When the flow rate is more than a certain value than the flow rate, an instruction is given to stop the liquid feeding by the liquid feeding means, and the measured flow rate is a flow rate that is smaller than the constant value by more than the flow rate of the liquid to be fed. The case is characterized in that the control is instructed to restart the liquid feeding by the liquid feeding means, and the liquid can be stably fed even at a minute flow rate of several μL to several tens μL per minute, Further, the time until the pressure becomes constant can be greatly shortened. Therefore, the liquid chromatograph can be easily “micro-sized” by providing the liquid chromatograph with the liquid feeding device using the liquid feeding method of the present invention.

It is a block diagram of a general liquid chromatograph. It is the figure which showed an example of the plunger pump and the suction / discharge operation | movement by the said pump. It is a top view of a reciprocating plunger pump (a) and a quick return plunger pump (b). It is a schematic diagram of plunger operation and pressure fluctuation in the reciprocating plunger pump (a) and quick return plunger pump (b). It is the figure which showed the plunger operation | movement and pressure fluctuation in a reciprocating type plunger pump (a) and a quick return type plunger pump (b) at the time of a high flow rate. It is the figure which showed the plunger operation | movement and pressure fluctuation in a reciprocating type plunger pump (a) and a quick return type plunger pump (b) at the time of a low flow rate. It is the figure which showed the plunger operation | movement and pressure fluctuation in a plunger pump when a cam is rotated at high speed at the time of liquid suction. It is the figure which showed another example of the plunger pump, and the suction / discharge operation | movement by the said pump. It is the figure which showed the plunger operation | movement and pressure fluctuation in the plunger pump of FIG. It is a block diagram which shows an example of a liquid chromatograph when using a syringe pump as a liquid feeding pump of an eluent. Furthermore, it is a block diagram which shows an example of a liquid chromatograph when a splitter is provided. It is a block diagram which shows an example of the liquid chromatograph which performs the feedback control by proportional control. It is the figure which showed the flow volume fluctuation | variation in the liquid chromatograph of FIG. It is a figure which shows the flowchart of the feedback control by proportional control. It is the figure which showed an example of the flow pattern and pressure fluctuation when accelerating the pressure rise at the time of a low flow rate. It is a flowchart which shows the liquid feeding method of this invention. It is a figure which shows an example of the relationship between the target flow volume and a drive flow volume in the liquid feeding method of this invention. It is a figure which shows an example of the relationship between the target flow volume and a drive flow volume in the liquid feeding method of this invention. It is the figure which showed typically the change of the flow volume in the liquid feeding method of this invention. It is the figure which showed typically the raise of the pressure at the time of a liquid feeding start with the liquid feeding method of this invention, and the conventional liquid feeding method which does not perform feedback control. It is the figure which showed an example of the liquid feeding apparatus using the liquid feeding method of this invention. It is the figure which showed typically the plunger position motor rotational speed and pressure fluctuation when the plunger pump shown in FIG. 8 was equipped in the liquid feeding apparatus shown in FIG. It is a block diagram of the liquid chromatograph used in the Example. It is the figure which showed the relationship between the target flow volume and drive flow volume in the liquid feeding method of this invention used in the Example. It is a figure which shows the result of Example 1 (A). a shows the change in the flow rate (output value of the thermal flow meter) with respect to the feeding time when the solution is fed by the conventional solution feeding method without feedback control, and b shows the feeding when the solution is fed by the solution feeding method of the present invention. It is the figure which showed the flow volume change with respect to liquid time, respectively. It is a figure which shows the result of Example 1 (A). a shows the pressure change with respect to the liquid feeding time when the liquid is fed by the conventional liquid feeding method without feedback control, and b shows the pressure change with respect to the liquid feeding time when the liquid is fed by the liquid feeding method of the present invention. It is a figure. It is the figure which expanded the liquid feeding start vicinity of the conditions 7 of Table 1 among the results of Example 1 (A). a is an enlarged view of the flow rate (output value of the thermal flow meter) change with respect to the liquid feeding time when the liquid is fed by the conventional liquid feeding method without feedback control, and b is the liquid feeding method according to the present invention. It is an enlarged view of the flow volume change with respect to the liquid feeding time at the time of doing. It is the figure which expanded the liquid feeding start vicinity of the conditions 7 of Table 1 among the results of Example 1 (A). a is an enlarged view of a pressure change with respect to a liquid feeding time when the liquid is fed by a conventional liquid feeding method without feedback control, and b is a pressure change with respect to a liquid feeding time when the liquid is fed by the liquid feeding method of the present invention. FIG. It is a figure which shows the result of Example 1 (B). a shows the change in the flow rate (output value of the thermal flow meter) with respect to the feeding time when the solution is fed by the conventional solution feeding method without feedback control, and b shows the feeding when the solution is fed by the solution feeding method of the present invention. It is the figure which showed the flow volume change with respect to liquid time, respectively. It is a figure which shows the result of Example 1 (B). a shows the pressure change with respect to the liquid feeding time when the liquid is fed by the conventional liquid feeding method without feedback control, and b shows the pressure change with respect to the liquid feeding time when the liquid is fed by the liquid feeding method of the present invention. It is a figure. In Example 1, it is the figure which put together the pressure change rate (magnitude | size of a pulsation) with respect to the flow volume. It is a figure which shows the chromatogram obtained in Example 2 (Eluent liquid supply amount: 3 microliters per minute). a is a chromatogram when the liquid is fed by the conventional liquid feeding method without feedback control, and b is a chromatogram when the liquid is fed by the liquid feeding method of the present invention. In Example 2, it is the figure which put together the reproducibility of the elution time with respect to a flow volume. It is the figure which showed the principle of the thermal type flow meter. FIG. 24 is a detailed view of a pressure sensor provided in the liquid chromatograph shown in FIG. 23.

  Hereinafter, the present invention will be described in more detail with reference to an eluent feeding device provided in a liquid chromatograph as an example, but these examples do not limit the present invention.

Example 1
In a liquid chromatograph, changes in flow rate and pressure when a minute flow rate of an eluent was sent were confirmed.

(A) Liquid feeding under low pressure conditions The apparatus shown in FIG. 23 was used as a liquid chromatograph. The liquid chromatograph shown in FIG. 23 includes an eluent (10), a liquid feed pump (1), a damper (26) / pressure sensor (2), a sample introduction valve (4), an analytical column (7), a detector (9 ) In series in the above order, and a flow meter (15) is provided between the pressure sensor (2) and the sample introduction valve (4), so that the flow rate of the eluent (10) by the liquid feed pump (1) is real-time. The monitor can be monitored with As the damper (26) / pressure sensor (2), an integrated type as shown in FIG. 35 was used. A pressure detector (33) is provided on one side of the diaphragm damper (26) provided with the diaphragm (32) and the buffer solution (31), and the eluent does not directly contact the pressure detector (33). The pressure sensor (2) used was KM15-S07 50PMA manufactured by Nagano Keiki. The liquid delivery pump (1) used the plunger pump shown in FIG. 8, the sample introduction valve (4) used a 2-position switching valve, and the sample loop (5) used a 0.5 μL capacity loop. As an analytical column (7) under low pressure conditions, TSKgel ODS-100V (inner diameter: 1.0 mm ID, length: 35 mm, particle diameter: 3 μm) manufactured by Tosoh was used. The detector (9) was Tosoh UV-visible detector UV-8020 (microcell) (detection wavelength: 254 nm), and the eluent (10) was a 60% acetonitrile aqueous solution. The flow meter (15) was a thermal type flow meter, LIQUID Mass Flow Meters L13 manufactured by Bronkhorst, which was used in a state where it was housed in a thermostat (17) controlled at 45 ° C. The actual flow rate by the liquid feeding pump (1) was calculated from the change in weight of the waste liquid from the detector (9) received in the measuring container (19) on the balance (18). The flow rate value obtained from the change in weight per hour by the balance includes the influence of temperature, error due to liquid flow, and the like. For this reason, it is difficult to accurately measure the absolute value, and a difference from the flow rate measurement value in the present invention may occur. Therefore, the flow rate value by the balance is only used as a reference value.

  In the liquid chromatograph shown in FIG. 23, when the liquid feeding method of the present invention is adopted, the analog output of the flow meter (15) is input to the flow rate determining means (16), and the programmable relay which is the flow rate determining means (16) The flow rate of the eluent (10) is controlled by sending an ON / OFF signal to the pump controller (3) and performing feedback control of Flow (stopping) / Stop (stopping) of the pump (1). The programmable relay as the flow rate determining means (16) is turned off when the control upper limit flow value is exceeded, and turned on when the control lower limit flow value is fallen below. The driving flow rate of the liquid feeding pump (1) is set to 38.6 μL / min when the target flow rate is 7 μL / min or less, and approximately 31.6 μL / min is added to the target flow rate value when the target flow rate is 7 μL / min or more. (FIG. 24).

表１に示す７つの条件で、フィードバック制御を行なう本発明の送液方法で送液した場合と、フィードバック制御を行なわない従来の送液方法で送液した場合とで、送液時間に対する流量変化（熱式流量計の出力値）を確認した結果を図２５に、送液時間に対する圧力変化を確認した結果を図２６に、それぞれ示す。また表１に示す条件の中で最も流量の少ない、条件７における送液開始時付近を拡大した図を図２７（流量変化）および図２８（圧力変化）に示す。

The flow rate change with respect to the liquid feeding time when the liquid is fed by the liquid feeding method of the present invention that performs feedback control under the seven conditions shown in Table 1 and when the liquid is fed by the conventional liquid feeding method that does not perform feedback control. FIG. 25 shows the result of confirming (the output value of the thermal flow meter), and FIG. 26 shows the result of confirming the pressure change with respect to the liquid feeding time. In addition, FIG. 27 (flow rate change) and FIG. 28 (pressure change) are enlarged views of the vicinity of the time when the liquid supply is started under the condition 7 with the smallest flow rate among the conditions shown in Table 1.

  When the liquid is fed by the conventional liquid feeding method without feedback control, the eluent is aspirated / discharged periodically by the liquid feeding pump, and the flow rate (the output value of the thermal flow meter) and the pressure are synchronized with it. Fluctuates periodically (FIGS. 25a, 26a, 27a and 28a). In addition, the flow rate decreases and the period increases. On the other hand, when the liquid is fed by the liquid feeding method of the present invention, the liquid feeding pump is driven at a flow rate about 30 μL / min higher than the target flow rate. Therefore, the flow / stop of liquid feeding by the liquid feeding pump is switched in a short time, and the suction operation by the liquid feeding pump is also performed at high speed, so that a drop in flow rate and pressure can be suppressed (FIGS. 25b, 26b, FIG. 27b and FIG. 28b).

  Further, when the liquid is fed by the conventional liquid feeding method, it takes a long time until the pressure becomes constant from the start of the liquid feeding, and particularly when the flow rate is low, this is remarkable (FIG. 26a). Specifically, it takes about 10 minutes until the pressure becomes constant (0.3 MPa) under the condition 7 in which the flow rate is the lowest among the conditions shown in Table 1 (FIG. 28a). On the other hand, in the case of liquid feeding by the liquid feeding method of the present invention, the liquid feeding pump is driven at a flow rate that is approximately 30 μL / min higher than the target flow rate, so the time until the pressure becomes constant is shortened. Specifically, under condition 7 in Table 1, the time required for the pressure to become constant (0.3 MPa) is about 1 minute (FIG. 28b).

(B) Liquid feeding under high pressure conditions The usefulness of the liquid feeding method of the present invention in the case of liquid feeding under higher pressure conditions than (A) was evaluated. The liquid chromatograph used was Example 1 except that TSKgel ODS-100V (inner diameter 0.3 mm ID, length 90 mm, particle size 3 μm) manufactured by Tosoh was used as the analytical column (7) under high pressure conditions. Is the same. Under this condition, a pressure of about 3 MPa, which is about 10 times that of Example 1, is generated near a flow rate of 3 μL / min.

  Under condition 7 in Table 1, the change in flow rate with respect to the liquid supply time (in the case of the liquid flow method of the thermal flow meter) when the liquid is supplied by the liquid supply method of the present invention and when the liquid is supplied by the conventional liquid supply method without feedback control. FIG. 29 shows the result of confirming the output value), and FIG. 30 shows the result of confirming the change in pressure with respect to the liquid feeding time. When liquid is fed by a conventional liquid feeding method, it takes about 50 minutes from the start of liquid feeding until the pressure becomes constant (3 MPa) (FIG. 30a). On the other hand, when the liquid is fed by the liquid feeding method of the present invention, the liquid feeding pump is driven at a flow rate about 30 μL / min higher than the target flow rate, so the time required for the pressure to become constant (3 MPa) is about 3 minutes. (FIG. 30b). That is, it can be seen that the effect of performing the liquid feeding method of the present invention appears more significantly under high pressure conditions.

  FIG. 31 shows the result of summarizing the pressure fluctuation rate (the magnitude of pulsation) after the pressure became constant in the liquid feeding performed in (A) and (B). Compared with the case where the liquid is fed by the liquid feeding method of the present invention, the liquid is fed by the conventional liquid feeding method which does not perform the feedback control, and is about 40% under the low pressure condition (A) and under the high pressure condition (B). Each can be reduced by about 80%. That is, when the liquid is fed by the liquid feeding method of the present invention, the pulsation is reduced, so that the actual flow rate fluctuation is reduced, and improvement in reproducibility of the elution time can be expected.

Example 2
The influence on the liquid chromatograph analysis by the liquid feeding method of the present invention was confirmed. The liquid chromatograph used is the same as that used in Example 1 (A). The eluent (10) was a 60% acetonitrile aqueous solution, and the sample (6) was a mixture of methyl p-hydroxybenzoate, butyl p-hydroxybenzoate and hexyl p-hydroxybenzoate. The amount of eluent (10) fed was 3 μL per minute (corresponding to condition 7 in Table 1). FIG. 32 shows the chromatogram obtained by analyzing the sample 10 times. 32, FIG. 32b shows a chromatogram when the liquid is fed by the liquid feeding method of the present invention, and FIG. 32a shows a chromatogram when the liquid is fed by the conventional liquid feeding method without feedback control.

  The amount of the eluent (10) to be fed is 15 μL / min (corresponding to condition 5 in Table 1), 7 μL / min (corresponding to condition 6 in Table 1), or 3 μL / min (corresponding to condition 7 in Table 1). Table 2 (15 μL / min) and Table 3 (7 μL / min) show the elution time of each component and the reproducibility of elution time (Cv [%]) obtained by analyzing the sample 10 times. ) And Table 4 (3 μL per minute). Further, FIG. 33 shows a plot of elution time reproducibility (Cv [%]) against the amount of eluent (10) fed.

フィードバック制御を行なわない従来の送液方法で送液した場合、流量が低くなるにつれて溶出時間の再現性（Ｃｖ［％］）が悪くなる傾向にある。特に毎分３μＬの微小流量で送液した場合、溶出時間の再現性（Ｃｖ［％］）は０．６７％から１．２％と極端に悪くなり、実際の分析では許容できないレベルとなっている。一方、本発明の送液方法で送液する場合、毎分３μＬから４０μＬまでの広い範囲で、溶出時間の再現性（Ｃｖ［％］）は０．２％程度を維持しており、微小流量を送液する液体クロマトグラフであっても、試料中に含まれる成分を再現性高く分析することができる。

When liquid is fed by a conventional liquid feeding method that does not perform feedback control, the reproducibility of elution time (Cv [%]) tends to deteriorate as the flow rate decreases. In particular, when the liquid is fed at a minute flow rate of 3 μL per minute, the reproducibility of elution time (Cv [%]) is extremely poor from 0.67% to 1.2%, which is unacceptable in actual analysis. Yes. On the other hand, when liquid is fed by the liquid feeding method of the present invention, the reproducibility of elution time (Cv [%]) is maintained at about 0.2% in a wide range from 3 μL to 40 μL per minute, Even in a liquid chromatograph that sends liquid, the components contained in the sample can be analyzed with high reproducibility.

1: Liquid feed pump 2: Pressure sensor 3: Pump controller 4: Sample introduction valve 5: Sample loop 6: Sample 7: Analytical column 8: Column thermostat 9: Detector 10: Eluent 11: Resistance tube 12: Branch block 13: Syringe pump 14: Flow path switching valve 15: Flow meter 16: Flow rate determination means 17: Constant temperature bath 18: Balance 19: Measuring container 20: Plunger 21: Check valve 22: Cam 23: Ball screw 24: Pump head 25 : Motor 26: Damper (pulsation removing device)
27: Flow rate determination unit 28: Temperature detection unit 29: Heater 30: Pipe 31: Buffer solution 32: Diaphragm 33: Pressure detection unit 34: Eluent passage unit

Claims (4)

A liquid feeding method for measuring the flow rate of the liquid fed by the liquid feeding means and performing liquid feeding control by the liquid feeding means based on the measured flow rate,
The liquid feeding control by the liquid feeding means instructs the liquid feeding means to feed a flow rate higher than the flow rate of the liquid to be fed by the liquid feeding means, and the measured flow rate should be the liquid feeding. When the flow rate is higher than the liquid flow rate by a certain value or more, the flow rate is instructed to stop by the liquid feeding means, and the measured flow rate is lower than the liquid flow rate to be fed by a certain value or more. When it becomes, the said liquid feeding method which is control which instruct | indicates to restart the liquid feeding by the said liquid feeding means. Based on the liquid feeding means for feeding the liquid, the flow rate measuring means for measuring the flow rate of the liquid fed by the liquid feeding means provided on the discharge side of the liquid feeding means, and the flow rate measured by the flow rate measuring means A liquid feeding device provided with a control means for performing liquid feeding control in the liquid feeding means,
A flow rate determination means for determining whether the flow rate measured by the flow rate measurement means is a flow rate greater than a certain value or less than a flow rate of the liquid to be delivered by the liquid delivery means;
The control means instructs the liquid feeding means to send a flow rate higher than the flow rate of the liquid to be fed by the liquid feeding means, and the flow rate measured by the flow rate measuring means is the flow rate of the liquid to be delivered. When the flow rate determination means determines that the flow rate is more than a certain value, the flow rate is measured by the flow rate measurement means, and the flow rate measured by the flow rate measurement means is greater than the flow rate of the liquid to be sent. If the flow rate determination means determines that the flow rate is less than a certain value, the liquid supply device is a means for instructing the liquid supply means to resume liquid supply. The liquid feeding device according to claim 2, wherein the flow rate measuring means is a thermal flow meter. A liquid feed section for feeding an eluent, a sample introduction section for introducing a sample, an analysis column section for separating each component in the introduced sample, and a detection section for detecting each component eluted from the analysis column section, The liquid chromatograph, wherein the liquid feeding unit is the liquid feeding device according to claim 2.

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