JP2011226914A - Liquid feeding device for liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid feeding device for liquid chromatography with two or more kinds of reaction reagents being added, the device enabling the reaction reagents to be easily mixed; allowing accurate liquid feeding on low flow rate region; and enabling a detector to obtain stabilized baseline.SOLUTION: The problem is solved by a liquid feeding device for liquid chromatography comprising pumps that feed two or more kinds of reagents, respectively; a mixing vessel that mixes and stores the reagents fed with the pump; and a pump that feeds the mixed reagent stored in the mixing vessel, and by liquid chromatography comprising the device.

Description

  The present invention relates to a liquid delivery device used in liquid chromatography. In particular, the present invention relates to an apparatus for feeding a reaction reagent used in reaction liquid chromatography and a liquid chromatography provided with the apparatus.

  Liquid chromatography is used for the separation and analysis of various sample components. When detecting or quantifying a sample component, a detector suitable for the component is used. The most frequently used are ultraviolet / visible absorption detectors. In this method, the sample component is detected or quantified based on the difference in ultraviolet / visible absorption between the sample component and the eluent. When the sample component does not have ultraviolet / visible absorption, a differential refractometer that performs detection or quantification based on the difference between the refractive index of the sample component and the refractive index of the eluent is used. However, because these detectors have low specificity, it may be difficult to detect or quantify only the target sample component, such as when there are many contaminants in the sample. In such a case, after separating the sample components with an analytical column, the sample components to be detected or quantified are mixed with a reagent that specifically reacts, derivatized by heating or the like, and then the derivatized sample components are ultraviolet / visible. There are methods for measuring absorption and fluorescence. This method is generally called post-column reaction liquid chromatography.

  FIG. 1 shows an embodiment of the flow path configuration of post-column reaction liquid chromatography. The sample (5) injected by the sample injection valve (3) is separated into each component by the analysis column (7). Reaction reagent A (11a) sent by the liquid feed pump (10a) and reaction reagent B (11b) sent by the liquid feed pump (10b) are added to the components eluted from the analytical column (7), Then, the reaction which produces | generates a derivative specific with respect to the component detected or quantified advances by passing through the inside of the reaction coil (8) provided in the reaction oven (9) temperature-controlled at suitable temperature. Thereafter, detection or quantification is performed by detecting the target component mainly with high sensitivity by the detector (12) that specifically detects the derivative.

  In general, the eluent (2) used in the post-column reaction liquid chromatography tends to have a low background to the detector (12), and the reaction reagent A (11a) and the reaction reagent B (11b) have a detector (12). ) Has a relatively high background. Therefore, when a change occurs in the composition of the mixture of the eluent (2) and the reaction reagent A (11a) and the reaction reagent B (11b) or the mixture of the reaction reagent A (11a) and the reaction reagent B (11b), The background of the detector (12) will fluctuate.

JP 2000-046819 A

  Several solutions have been studied as solutions to the above-mentioned problems. For example, when adding the reaction reagent A (11a) and the reaction reagent B (11b) to the component eluted from the analytical column (7), the reaction reagent A (11a) and the reaction reagent B (11b) are first combined. Then, after passing through the mixing coil (25), the reaction reagent mixture is homogenized to some extent, and then added to the components eluted from the analytical column (7) to cause a reaction (see FIG. 2).

  Patent Document 1 discloses that the elution liquid supply pump and the reaction reagent liquid supply pumps are all controlled by a signal based on a single clock signal so that the liquid supply period is regularized. And a method for suppressing fluctuations in the composition of the reaction reagent. However, since the eluent feed pump and the reaction reagent feed pump normally use the plunger pump shown in FIG. 3, it is difficult to make the composition uniform.

  The plunger pump (FIG. 3) mainly includes a pump head (24), a plunger (16), and inlet / outlet check valves (18a, 18b). By pulling the plunger (16) (moving leftward in FIG. 3), liquid is sucked into the chamber (17) via the inlet check valve (18a) (FIG. 3a), and the plunger (16) is moved. By pushing (moving rightward in FIG. 3), the liquid is discharged from the chamber (17) through the outlet check valve (18b). Usually, in order to improve the stability of liquid feeding, control is performed so that the suction operation is completed in as short a time as possible and the discharge operation is performed at a constant speed.

  In post-column reaction liquid chromatography, two types of reaction reagents are often used, and two pumps for feeding the reaction reagents are also required. Therefore, when a plunger pump is used as a reaction reagent liquid feed pump, the liquid feed pattern is, for example, as shown in FIG. FIG. 4 shows a flow rate pattern when the flow rates of the reaction reagent A and the reaction reagent B are the same (specifically, the flow rates of the reaction reagent A and the reaction reagent B are both 20 μL / min). When both the flow rates of the reaction reagent A and the reaction reagent B are the same, theoretically, the flow rate pattern when added together has the same period as shown in FIG. 4c. However, when the flow rates of reaction reagent A and reaction reagent B are slightly different as shown in FIG. 5 (specifically, the flow rate of reaction reagent A is 20 μL / min and the flow rate of reaction reagent B is 19 μL / min), When this is done, the cycle of the flow velocity pattern is not constant (FIG. 5c). If there is no periodicity in the liquid feeding pattern when combined, uniform mixing becomes extremely difficult even if mixing is performed by the subsequent mixing coil, resulting in a loss of stability of the detector and a decrease in detection accuracy. . Further, even if the cycle of the liquid feeding pattern when the summation is performed as shown in FIG. 4c, a mixing coil having a capacity several times the cycle of the pump is required to make the composition of the mixed reagent completely constant. . In the case of a plunger pump (FIG. 3), the plunger volume (i.e., the volume of the chamber (17)) is typically 50 μL to several hundred μL, and the pump cycle increases as the flow rate decreases. Therefore, when the flow rate of the reaction reagent is relatively high, the method of adjusting the pump cycle is effective to some extent, but when the flow rate of the reaction reagent is low, the method of adjusting the pump cycle is insufficient and mixing is insufficient. There is a problem that impairs sex.

  Next, the case where the electromagnetic diaphragm pump shown in FIG. 6 considered to be suitable for feeding a low flow rate is used as the reaction reagent delivery pump will be examined. In the electromagnetic diaphragm pump (FIG. 6), when the electromagnetic coil (28) is energized / cut off, the cylindrical piston (27) moves up and down, and the diaphragm (26) contracts / expands in conjunction with the vertical movement. This makes it possible to send liquids. The diaphragm (26) is provided with two ports (for liquid inlet and outlet), and each port is provided with a simple check valve (29a, 29b). Thereby, the liquid feeding by contraction / expansion of the diaphragm (26) is controlled in one direction. The flow rate is determined by the stroke of the piston (27), and the stroke is adjusted by limiting the return position of the piston (27) by adjusting the stroke adjustment stopper (30). When the electromagnetic diaphragm pump is applied to a reaction reagent feeding pump in post-column reaction liquid chromatography using two kinds of reaction reagents, for example, the reaction reagent A (11a) and the reaction reagent have the flow path configuration shown in FIG. B (11b) can be sent alternately and in the same cycle, and the two reagents can be mixed easily. FIG. 8 shows a discharge pattern of a pump for feeding each reaction reagent when the flow rates of the reaction reagent A and the reaction reagent B are the same. In the case of the electromagnetic diaphragm pump shown in FIG. 6, the flow rate can be adjusted by adjusting the stroke adjustment stopper (30) and adjusting the return position of the piston (27) even if the two reagent delivery amounts are different. Therefore, the pump A for feeding the reaction reagent A and the pump B for feeding the reaction reagent B are synchronized with each other (so that the discharge pattern shown in FIG. 9 is achieved). Thus, the two reagents can be easily mixed.

  In the case where the setting is such that the reaction reagent A and the reaction reagent B are alternately discharged using an electromagnetic diaphragm pump, if the flow path configuration has no load on the downstream side of the pump as shown in FIG. The liquid flows in an equal amount and regularly in the order of reagent A-reagent B-reagent A-reagent B-reagent A-reagent B (FIG. 13a). From the above, the electromagnetic diaphragm pump seems to be useful as a reaction reagent feed pump for post-column reaction liquid chromatography.

  However, in the electromagnetic diaphragm pump, the piston is driven by energizing / cutting off the electromagnetic coil, and since the diaphragm is usually made of resin, the pressure resistance is 0.2 MPa or less and the plunger pump (FIG. 3). ) Is extremely low. Therefore, when the load pressure increases, the flow rate obtained by the electromagnetic diaphragm pump varies greatly. In other words, even with the same setting, there is a problem that the actual flow velocity is greatly reduced as the load increases (FIG. 10). Therefore, in the case of a flow path configuration having a load on the downstream side of the pump as shown in FIG. 12, the reaction reagent A (11a) and the reaction reagent B (11b) are respectively used as the electromagnetic diaphragm pumps (10a, 10b). When trying to supply continuously, the specified flow rate cannot be obtained. When the flow rate characteristics with respect to the load pressure are different between the pump A (10a) for feeding the reaction reagent A and the pump B (10b) for feeding the reaction reagent B, as shown in FIG. 13b, May flow non-uniformly or, in some cases, either of the reaction reagents may not flow at all. Therefore, when an electromagnetic diaphragm pump is applied as a reaction reagent feed pump in post-column reaction liquid chromatography, it is difficult to provide liquid chromatography with excellent detection and quantification accuracy by simply applying it.

  Therefore, an object of the present invention is to easily mix the reaction reagents in a liquid chromatography to which two or more kinds of reaction reagents are added, enable accurate liquid feeding in a low flow rate range, and provide a stable base in the detector. It is providing the liquid feeding apparatus from which a line is obtained. In addition, the present invention provides an apparatus that can be used as a reaction reagent liquid feeding pump by making up for the low pressure resistance while taking advantage of the characteristics of an electromagnetic diaphragm pump that can easily control the liquid suction / discharge cycle. With the goal. Still another object of the present invention is to provide a liquid chromatography equipped with such a liquid delivery device.

  The present invention made to achieve the above object includes the following inventions.

The first invention is
A liquid feeding device for liquid chromatography that mixes two or more kinds of reagents and feeds the mixed reagents,
A pump for feeding each of the two or more types of reagents;
A mixing container for mixing and storing the reagent fed by the pump;
A pump for feeding the mixed reagent stored in the mixing container;
It is a liquid-feeding apparatus for liquid chromatography provided with.

  The second invention is a liquid feeding device according to the first invention, wherein the pump for feeding two or more kinds of reagents and / or the pump for feeding the mixed reagent stored in the mixing container is an electromagnetic diaphragm pump. It is.

The third invention is
Mixing container
A port that accepts two or more reagents,
A port for feeding the stored mixed reagent;
Reagent discharge port to keep the mixed reagent storage capacity constant,
It is a liquid feeding apparatus as described in 1st or 2nd invention which is a mixing container provided with.

The fourth invention is
Introducing means for introducing the sample;
An analytical column for separating the sample introduced from the introduction means;
Detection means for detecting or quantifying components eluted from the analytical column;
A liquid chromatography comprising:
It is a liquid chromatography provided with the liquid sending device according to the first to third inventions between the upstream side of the analytical column or the downstream side of the analytical column and the detection means.

The fifth invention is
A liquid feeding device is provided between the downstream side of the analytical column and the detection means,
A reaction means for reacting the eluted component and the mixed reagent between a position where the mixed reagent sent by the liquid delivery device is added to the component eluted from the analytical column and a detection means; It is a liquid chromatography as described in 4th invention.

  Hereinafter, the present invention will be described in detail.

The liquid delivery device of the present invention is
A pump for feeding two or more reagents,
A mixing container for mixing and storing the reagent fed by the pump;
A mixed reagent feeding pump for sucking and discharging the mixed reagent stored in the mixing container;
The liquid delivery device is characterized by pre-column reaction liquid chromatography in which the sample components are derivatized and analyzed before the sample is introduced into the analytical column, or the sample components eluted from the analytical column are derivatized and analyzed. It can be used as an apparatus for adding a reagent for derivatizing a sample component in the post-column reaction liquid chromatography to be performed.

  The pump for supplying two or more kinds of reagents and the mixed reagent feeding pump provided in the liquid feeding device of the present invention may be any pump that is usually used in liquid chromatography, and examples include a plunger pump and an electromagnetic diaphragm pump. However, when the amount of reagent to be fed is small, an electromagnetic diaphragm pump capable of stable feeding in a low flow rate region is used as a pump for feeding two or more reagents and / or a mixed reagent feeding pump. And preferred. The electromagnetic diaphragm pump transmits the reciprocating motion of a piston driven and controlled by an electromagnetic coil (also referred to as a solenoid) to a diaphragm chamber provided in the flow path via a check valve, thereby supplying liquid feeding force. Any liquid feed pump having the operation principle to be obtained may be used. In the electromagnetic diaphragm pump, the suction time and the discharge time, that is, the liquid feeding cycle can be freely set within the specification range by the control pulse signal, and the flow rate can be set by adjusting the stroke of the piston.

As one aspect of the mixing container provided in the liquid feeding device of the present invention,
A port for receiving two or more kinds of reagents sent by each pump;
A port for sucking and feeding the mixed two or more kinds of reagents with a mixed reagent feed pump;
A reagent discharge port (overflow port) for keeping the volume of the two or more types of mixed reagents constant;
Can be mentioned. In addition, when using the liquid feeding apparatus of this invention as an apparatus which adds the reagent for derivatizing the sample component in precolumn reaction liquid chromatography, it introduce | transduces into the said mixing container by a sample introduction means as shown in FIG. A port for receiving the prepared sample may further be provided.

  Hereinafter, the present invention will be described in detail with reference to the drawings.

One embodiment of the liquid delivery device of the present invention is shown in FIG. 14a and 14b are both devices for adding a reaction reagent for derivatizing components eluted from the analytical column in post-column reaction liquid chromatography, and for supplying reaction reagent A (11a). Pump (10a) for
A reaction reagent B (11b) pump (10b) for feeding;
A mixing vessel (20);
And a mixed reaction reagent solution pump (19). FIG. 14a shows a mode in which the reaction reagent A (11a) sent by the pump (10a) and the reaction reagent B (11b) sent by the pump (10b) are individually introduced into the mixing container (20). FIG. 14b shows that the reaction reagent A (11a) sent by the pump (10a) and the reaction reagent B (11b) sent by the pump (10b) are combined in advance and mixed in the mixing container (20). The supplied modes are shown respectively. In the liquid delivery device of FIG. 14, in order to stabilize the mixing ratio of the reaction reagent A (10a) and the reaction reagent B (10b), the mixing container provided in a state almost at atmospheric pressure (that is, a state with almost no load). (20) After supplying the reaction reagent A (11a) and the reaction reagent B (11b) to each other using separate pumps (10a, 10b) and mixing them, the mixed reaction reagents are fed at a constant cycle using the pump (19). Pump the liquid. The fed mixed reaction reagent is mixed with the sample components eluted from the analysis column, and the components are derivatized in a reaction means such as a reaction coil (8). The mixing container (20)
One or more ports (preferably two ports as shown in FIG. 14a) for receiving reaction reagent A (11a) and reaction reagent B (11b);
A port for feeding the stored mixed reaction reagent with a pump (19);
A mixed reaction reagent discharge port (overflow port, 23) for keeping the mixed reaction reagent storage capacity constant;
It has. When the amount of reaction reagent to be fed is small, it is preferable that the pump (10a), the pump (10b), and the pump (19) are all electromagnetic diaphragm pumps.

  When the reaction reagent A liquid delivery pump (10a) and the reaction reagent B liquid delivery pump (10b) are electromagnetic diaphragm pumps, the pump (10a) and the pump (10b) repeat the suction / discharge in a cycle of several seconds. It is preferable to send the solution to the mixing container (20). The mixed reaction reagent is homogenized in the mixing container (20), and a part of the mixed reaction reagent is fed by the mixed reaction reagent feeding pump (19) and added to the components in the sample eluted from the analytical column. . The mixed reaction reagent exceeding the capacity of the mixing container (20) is discharged out of the container through the discharge port (overflow port, 23). FIG. 15 shows an enlarged view of the vicinity of the mixing container (20) in the liquid delivery device of FIG. The amount (flow rate) Fm of the mixed reaction reagent is set slightly smaller than the sum of the flow rate Fa of the reaction reagent A and the flow rate Fb of the reaction reagent B. Thereby, the amount of the mixed reaction reagent in the mixing container gradually increases to the container capacity and finally reaches the same capacity as the container capacity. Thereafter, the increment is discarded outside the system from the mixed reaction reagent discharge port (overflow port) (flow rate: Fw). 15a is an enlarged view of the vicinity of the mixing container (20) of FIG. 14a, and FIG. 15b is an enlarged view of the vicinity of the mixing container (20) of FIG. 14b. In order to improve the mixing efficiency of the reaction reagent A and the reaction reagent B supplied to the mixing container (20), a stirrer (21) is placed in the mixing container (20), and the drive installed outside. The reaction reagent may be stirred by rotating the stirring bar (21) by the unit (22). The difference between the total flow rate Fa of the reaction reagent A and the flow rate Fb of the reaction reagent B (ie, Fa + Fb) and the flow rate Fm of the mixed reaction reagent (ie, Fa + Fb−Fm) is preferably as small as possible. This is because the difference becomes overflow Fw and is discarded.

Fa + Fb> Fm
Fw = Fa + Fb-Fm
Flow rate of reaction reagent A: Fa, flow rate of reaction reagent B: Fb
Mixed reaction reagent flow rate: Fm, overflow flow rate: Fw
In the liquid feeding device of the present invention, no load is applied to the reaction reagent A liquid feeding pump and the reaction reagent B liquid feeding pump that determine the composition of the mixed reaction reagent. For this reason, the amount of the reaction reagent fed can always be kept at a constant value, and the mixing ratio can be kept constant. Further, the mixed reagent feed pump is subjected to a slight load by a reaction means such as a reaction coil and a detection means existing downstream, but the fluctuation of the load is small and the liquid feed amount is stable (see FIG. 16).

  The change in the composition of each reaction reagent in the mixing container was calculated numerically when the flow rate when the reaction reagent A and the reaction reagent B were supplied to the mixing container and the capacity of the mixing container were assumed. FIG. 17 shows the composition of each reaction reagent in the container when the volume of the mixing container is changed assuming that the flow rate Fa of the reaction reagent A is 10 μL / min and the flow rate Fb of the reaction reagent B is 10 μL / min. FIG. 17a shows a container capacity of 500 μL, FIG. 17b shows a container capacity of 200 μL, FIG. 17c shows a container capacity of 100 μL, and FIG. Shows the composition change when the container capacity is assumed to be 50 μL, and FIG. As time passes, the ratio of the reaction reagent B to the mixed reaction reagent in the mixing container increases, and finally the ratio of the reaction reagent A and the reaction reagent B becomes constant at 50%. For example, when the volume of the mixing container is 50 μL, it is stabilized in about 10 minutes. The larger the mixing container capacity, the longer it takes to reach 50%. The larger the mixing container capacity, the better the mixing of the reaction reagent. However, when the amount of reaction reagent A and / or reaction reagent B is intentionally changed, the composition of each reaction reagent actually changes. Takes time. In consideration of the possibility that the measurement result may fluctuate due to deterioration of the mixed reagent or the like, it is preferable that the volume of the mixing container is as small as possible.

The liquid chromatography apparatus for liquid chromatography of the present invention comprises:
A pump for feeding two or more reagents,
A mixing container for mixing and storing the reagent fed by the pump;
A pump for feeding the mixed reagent stored in the mixing container;
It is characterized by the fact that it is equipped with a mixing container that does not apply excessive back pressure, and the pump that sends two or more types of reagent and the pump that sends the mixed reagent stored in the mixing container are loaded. It hardly takes. For this reason, the electromagnetic diaphragm pump, which has been difficult to apply because of its low pressure resistance, is used as a pump for feeding two or more types of reagents and / or a pump for feeding mixed reagents stored in a mixing container. It becomes possible to apply. The electromagnetic diaphragm pump is easy to control the liquid suction / discharge cycle, and even if the amount of each reagent is the same or different, the amount of liquid can be changed by adjusting the return position of the piston. Since it is possible to adjust the solution feeding cycle to be synchronized, the reaction reagents can be mixed reliably. Therefore, by using an electromagnetic diaphragm pump as the pump for feeding two or more types of reagents and / or the pump for feeding the mixed reagent stored in the mixing container provided in the liquid feeding device of the present invention, a particularly low flow rate. The liquid feeding stability in the region can be accurately controlled. In addition, when the reagent discharge port (overflow port) for keeping the volume of the mixed reagent constant is provided in the mixing container provided in the liquid feeding device of the present invention, the suction of the pump for feeding the mixed reagent stored in the mixing container Since air can be prevented from being mixed into the pipe, the mixing ratio obtained when the flow rate of each reagent is changed can be predicted.

  The liquid delivery device of the present invention is a device for adding a reagent for derivatizing a sample component in a precolumn reaction liquid chromatography in which the sample component is derivatized and analyzed before the sample is introduced into the analytical column, or eluted from the analytical column. As a device for adding a reagent for derivatizing a sample component in post-column reaction liquid chromatography in which the sample component is derivatized and analyzed, it can be provided in liquid chromatography.

It is the figure which showed one aspect | mode (in the case of introduce | transducing a some reaction reagent directly to a reaction system) of the flow-path structure of a post column reaction liquid chromatography. It is the figure which showed another aspect (When introduce | transducing into a reaction system after mixing a some reaction reagent beforehand) of the flow-path structure of a post column reaction liquid chromatography. It is the figure which showed the structure of the general plunger pump. a shows the step of sucking the liquid, and b shows the step of discharging the liquid. It is the figure which showed an example (when the flow rate of the reaction reagent A and the reaction reagent B corresponds completely) when feeding 2 types of reaction reagents using each plunger pump. a shows the flow rate pattern of reaction reagent A, b shows the flow rate pattern of reaction reagent B, and c shows the total flow rate pattern of reaction reagent A and reaction reagent B, respectively. It is the figure which showed another example (when the flow rate of the reaction reagent A and the reaction reagent B is slightly different) when the two types of reaction reagents are sent using the respective plunger pumps. a shows the flow rate pattern of reaction reagent A, b shows the flow rate pattern of reaction reagent B, and c shows the total flow rate pattern of reaction reagent A and reaction reagent B, respectively. It is the figure which showed the structure of the general electromagnetic diaphragm pump. a shows the step of sucking the liquid, and b shows the step of discharging the liquid. It is the figure which showed the one aspect | mode of the flow-path structure when an electromagnetic diaphragm pump is used as a pump which sends 2 types of reaction reagents. It is the figure which showed an example (when the flow rate of the reaction reagent A and the reaction reagent B corresponds completely) when sending 2 types of reaction reagents using each electromagnetic diaphragm pump. a shows the discharge pattern of the pump A that sends the reaction reagent A, b shows the discharge pattern of the pump B that sends the reaction reagent B, and c shows the total discharge pattern of the pump A and pump B, respectively. It is the figure which showed another example (when the flow rates of the reaction reagent A and the reaction reagent B differ) when two types of reaction reagents are sent using the respective electromagnetic diaphragm pumps. FIG. A shows the discharge pattern of the pump A that sends the reaction reagent A, FIG. B shows the discharge pattern of the pump B that sends the reaction reagent B, and FIG. C shows the total discharge pattern of the pump A and pump B. . It is the figure which showed an example of the flow volume characteristic with respect to load when sending liquid using a general electromagnetic diaphragm pump. It is the figure which showed the flow of each reagent when two types of reaction reagents were sent using each electromagnetic diaphragm pump in the flow-path structure without a load in the downstream of a pump. a shows the step of discharging the reaction reagent A by the pump A, and b shows the step of discharging the reaction reagent B by the pump B. It is the figure which showed the flow of each reagent when two types of reaction reagents were sent using each electromagnetic diaphragm pump in the flow-path structure with a load in the downstream of a pump. a shows the step of discharging the reaction reagent A by the pump A, and b shows the step of discharging the reaction reagent B by the pump B. FIG. 13 is a schematic diagram of the flow of each reagent when two types of reaction reagents are fed using respective electromagnetic diaphragm pumps in the flow path configuration shown in FIGS. 11 and 12. a shows the flow in the channel configuration shown in FIG. 11, and b shows the flow in the channel configuration shown in FIG. It is the figure which showed the one aspect | mode of the liquid feeding apparatus of this invention. a is a mode in which two types of reaction reagents sent by each pump are directly introduced into the mixing container, and b is a mode in which two types of reaction reagents sent by the respective pumps are mixed in advance and then introduced into the mixing container. Each embodiment is shown. It is the figure which expanded the mixing container vicinity among the liquid feeding apparatuses of FIG. 14A is an enlarged view of the vicinity of the mixing container of the liquid delivery device of FIG. 14A, and FIG. 14B is an enlarged view of the vicinity of the mixing container of the liquid delivery device of FIG. 14B. It is the figure which showed operation | movement of the reaction reagent A liquid feed pump (pump A), the reaction reagent B liquid feed pump (pump B), and the mixed reaction reagent liquid feed pump when using the liquid delivery apparatus of this invention. It is the figure which showed the composition change in a mixing container when the liquid feeding apparatus of this invention is used. The flow rates of reaction reagent A (Fa) and reaction reagent B (Fb) are 10 μL per minute, a is a mixing container volume of 500 μL, b is a mixing container volume of 200 μL, c is a mixing container volume of 100 μL, and d is The mixing container capacity is 50 μL, and e indicates the case where the mixing container capacity is 20 μL. FIG. 3 is a diagram showing a flow channel configuration of a liquid chromatography provided with the liquid feeding device of the present invention used in Example 1. 1 is a diagram showing a derivatization reaction of glucose used in Example 1. FIG. 2 is a chromatogram when glucose is measured by liquid chromatography used in Example 1. FIG. FIG. 5 is a diagram showing operations of a reaction reagent A liquid feed pump (pump A), a reaction reagent B liquid feed pump (pump B), and a mixed reaction reagent liquid feed pump in the measurement of Example 1. It is the figure which showed the flow-path structure of the apparatus provided with the liquid feeding apparatus of this invention used in Example 2. FIG. FIG. 5 is a diagram showing operations of a reaction reagent A liquid feeding pump, a reaction reagent B liquid feeding pump, and a mixed reaction reagent liquid feeding pump in the measurement of Example 2. a shows the operation of the reaction reagent A solution pump, b shows the operation of the reaction reagent B solution pump, and c shows the operation of the mixed reaction reagent solution pump. FIG. 6 is a diagram showing the results obtained in Example 2. a shows the change in the output of the ultraviolet detector, and b shows the actual change in the flow rate of the mixed reaction reagent. It is the figure which showed the one aspect | mode of the flow-path structure when the liquid feeding apparatus of this invention is applied to a precolumn reaction liquid chromatography.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
In order to demonstrate the effect of the liquid delivery device of the present invention, sugar analysis was performed using a post-column reaction liquid chromatography equipped with the liquid delivery device of the present invention.

  Aldol sugars are converted to substances (derivatives) having ultraviolet absorption at 280 nm to 300 nm by heating with 2-cyanoacetamide in the presence of strong alkali (see FIG. 19). The sugar analysis is performed by detecting the converted derivative with a detector.

  FIG. 18 shows the flow path configuration of the post-column reaction liquid chromatography used in this example. The sample (5) injected from the sample injection valve (3) is transferred to the analysis column (7) by the eluent (2) sent by the eluent feed pump (1), and each component is subjected to normal phase chromatography. Separated. For each separated component, a 2.5% 2-cyanoacetamide solution (reaction reagent A (11a)) and a 0.1 M NaOH solution (reaction reagent B (11b)) were mixed / The solution was fed and continuously added in a state heated to 87 ° C. by a preheating coil (32), and then a derivative was produced in a reaction oven (9) kept at 45 ° C., and the product was detected by an ultraviolet detector (12 ). Tosoh CCPMII is used as the eluent feed pump (1), Tosoh UV-8020 (microcell, wavelength 300 nm) is used as the detector (12), and Tosoh CO-8020 is used as the column oven (9). As a reagent and mixed reaction reagent feed pump (10a, 10b, 19), a solenoid-driven diaphragm feed pump FMM20 manufactured by KNF was used. A resistance tube (31a) is disposed downstream of the detector for the purpose of preventing the generation of bubbles due to heating so that a slight pressure is applied to the detector. A resistance tube (31b, 31c) is disposed downstream of the electromagnetic diaphragm pump (on the discharge unit side) for the purpose of suppressing the actual liquid supply amount and for the purpose of relaxing the pulsating flow. A glass container having a capacity of about 300 μL was used as the mixing container (20). The eluent (2) is acetonitrile / water (75/25) containing 20 mM triethylamine, and the analytical column (7) is a normal phase chromatography column (manufactured by Tosoh Corporation, TSKgel Amide-80, particle size: 5 μm, inner diameter: 1 mm). , Length: 200 mm), and the eluent was fed at 27 μL per minute using the eluent feed pump (1). The reaction coil (8) was a PTFE tube having an inner diameter of 0.2 mm and a length of 2 m. As the sample (5), glucose (concentration 25 μg / mL) was used, and 0.5 μL was introduced into the analytical column (7) using the sample injection valve (3).

  Electromagnetic diaphragm pump (10a) for feeding reaction reagent A (2-cyanoacetamide solution, 11a), reaction reagent B (0.1M NaOH solution, 11b) and a mixture of reaction reagent A and reaction reagent B Table 1 shows the suction / discharge cycle (setting value) and the actual flow velocity (actual measurement) of 10b and 19). In addition, FIG. 21 shows a liquid feeding pattern (control value) in this example.

前記条件で得られたクロマトグラムを図２０に、グルコースの溶出時間と面積、高さの再現性（ｎ＝１０）をまとめた表を表２にそれぞれ示す。溶出時間のＣＶが０．２６％、面積のＣＶが４．５％と良好な値を示しており、２つの反応試薬が安定的に送液されていることを示唆している。

A chromatogram obtained under the above conditions is shown in FIG. 20, and a table summarizing glucose elution time, area and height reproducibility (n = 10) is shown in Table 2. The CV of elution time was 0.26%, and the CV of area was 4.5%, indicating good values, suggesting that the two reaction reagents were stably fed.

実施例２
本発明の送液装置の効果を実証するため、紫外吸収を有する液体と紫外吸収を有しない液体（純水）を送液し、送液安定性、混合効率の検証を実施した。

Example 2
In order to verify the effect of the liquid feeding device of the present invention, a liquid having ultraviolet absorption and a liquid not having ultraviolet absorption (pure water) were fed, and liquid feeding stability and mixing efficiency were verified.

  FIG. 22 shows the flow path configuration of the apparatus used in this example. Using pure water as the reaction reagent A (11a) and 1% acetone aqueous solution as the reaction reagent B (11b), and using an electromagnetic diaphragm pump (10a, 10b), the solution is sent to the mixing container (20) in a cycle of 4 seconds. did. Further, the mixed solution in the mixing container (20) is fed at a cycle of 10 seconds by another electromagnetic diaphragm pump (19), and the change in the composition is monitored by an ultraviolet detector (12) (detection wavelength: 254 nm). did. Resistance pipes (31b, 31c) are provided downstream for the purpose of suppressing pulsation of the electromagnetic diaphragm pumps (10a, 10b). A UV-Vis Spectrometer Detector (Spectra Academy) manufactured by K-Mac was used as the ultraviolet detector (12), and a solenoid-driven diaphragm feed pump FMM20 manufactured by KNF was used as the electromagnetic diaphragm pump (10a, 10b, 19). . Downstream of the ultraviolet detector (12), an electronic balance (34) and a flow rate calculator (35) for integrating the flow rate from time and weight increase are provided for the purpose of measuring the actual flow rate in real time. The mixing container (20) was a glass container having a capacity of about 300 μL.

  The liquid feeding pattern (control value) in a present Example is shown in FIG. In FIG. 24, only reaction reagent B was sent from 0 to 120 minutes, only reaction reagent A was sent from 120 to 270 minutes, and reaction reagent A and reaction reagent B were sent from 270 to 350 minutes. It shows the actual measured flow rate and change in UV absorption. As can be seen from a comparison between FIG. 23c and FIG. 24b, although the suction / discharge operation by the electromagnetic diaphragm pump is intermittently performed, the actual flow rate is almost constant and shows a continuous value. This is because the suction / discharge cycle of the electromagnetic diaphragm pumps (10a, 10b, 19) is fast, and the resistance pipe (31a) installed downstream reduces the pulsating flow. Further, as can be seen from a comparison between FIG. 24a and FIG. 24b, it can be seen that even if the composition of the solution to be finally fed changes, the actual flow rate hardly changes and is constant.

Example 3
One mode of the precolumn reaction liquid chromatography provided with the liquid feeding device of the present invention is shown in FIG. In the liquid chromatography shown in FIG. 25, the mixing container (20) provided in the liquid delivery device of the present invention includes:
A port for receiving the sample (5) introduced by the sample introduction means (36);
A port for receiving the reaction reagent A (11a) sent by the reaction reagent A feeding pump (10a);
A port for receiving the reaction reagent B (11b) sent by the reaction reagent B feeding pump (10b);
A port for feeding a solution in which the reaction reagent A, the reaction reagent B and the sample are mixed with a liquid feed pump (19);
A reagent discharge port (23) for keeping the volume of the mixed solution of reaction reagent A, reaction reagent B and sample constant;
Is provided.

The sample (5) introduced by the sample introduction means (36) is introduced into the mixing container (20) and mixed with the reaction reagent A (11a) and the reaction reagent B (11b) in the mixing container (20). The mixed solution passes through a reaction coil (8) provided in a reaction oven (9) by a liquid feed pump (19) to perform a derivatization reaction of the sample component. The derivatized sample component is introduced into the liquid chromatography sample injection valve (3) and introduced into the analytical column (7) by switching the injection valve (3) for specific detection.

1: Eluent feed pump 2: Eluent 3: Sample injection valve 4: Sample loop 5: Sample 6: Column oven 7: Analysis column 8: Reaction coil 9: Reaction oven 10: Reaction reagent feed pump 11: Reaction reagent 12: Detector 13: Waste liquid 14: Cam 15: Cam rotation center 16: Plunger 17: Chamber 18: Check valve 19: Mixing reaction reagent feed pump 20: Mixing container 21: Stirrer 22: Mixer (drive unit)
23: Reagent discharge port 24: Pump head 25: Mixing coil 26: Diaphragm 27: Piston 28: Electromagnetic coil 29: Check valve of electromagnetic pump 30: Stroke adjustment stopper 31: Resistance pipe 32: Preheating coil 33: Cap 34 : Electronic balance 35: Flow rate calculator 36: Sample introduction means

Claims (5)

A liquid feeding device for liquid chromatography that mixes two or more kinds of reagents and feeds the mixed reagents,
A pump for feeding each of the two or more types of reagents;
A mixing container for mixing and storing the reagent fed by the pump;
A pump for feeding the mixed reagent stored in the mixing container;
A liquid-feeding device for liquid chromatography, comprising: The liquid feeding device according to claim 1, wherein the pump for feeding two or more kinds of reagents and / or the pump for feeding the mixed reagent stored in the mixing container is an electromagnetic diaphragm pump. Mixing container
A port that accepts two or more reagents,
A port for feeding the stored mixed reagent;
Reagent discharge port to keep the mixed reagent storage capacity constant,
The liquid feeding device according to claim 1, wherein the liquid feeding device is a mixing container. Introducing means for introducing the sample;
An analytical column for separating the sample introduced from the introduction means;
Detection means for detecting or quantifying components eluted from the analytical column;
A liquid chromatography comprising:
4. A liquid chromatography comprising the liquid feeding device according to claim 1 between the upstream side of the analytical column or the downstream side of the analytical column and the detection means. A liquid feeding device is provided between the downstream side of the analytical column and the detection means,
The apparatus further comprises reaction means for reacting the eluted component with the mixed reagent between a position where the mixed reagent sent by the liquid delivery device is added to the component eluted from the analytical column and the detection means. Item 5. Liquid chromatography according to Item 4.

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