WO2009151096A1

WO2009151096A1 - Liquid chromatograph and gradient liquid feed system

Info

Publication number: WO2009151096A1
Application number: PCT/JP2009/060686
Authority: WO
Inventors: 徹夏目; 洋中山
Original assignee: 独立行政法人産業技術総合研究所; 独立行政法人理化学研究所
Priority date: 2008-06-13
Filing date: 2009-06-11
Publication date: 2009-12-17
Also published as: JPWO2009151096A1

Abstract

Provided is a liquid chromatograph equipped with a gradient liquid feed system which can feed liquid at an extremely low flow rate of several nano liters per minute stably with high precision. Provided is a gradient liquid feed system (2) performing liquid feed by forming an eluate gradient (G) in a single stroke syringe pump. Also provided is a liquid chromatograph (1) comprising the gradient liquid feed system (2) and provided, in the upstream of a column (14) for separating the components of a sample, with an injection port (15) for introducing the eluate gradient (G) fed from the gradient liquid feed system (2) into a channel (145).

Description

Liquid chromatograph and gradient liquid feeder

The present invention relates to a liquid chromatograph and a gradient liquid feeding apparatus. More specifically, the present invention relates to a gradient liquid feeding device that forms a gradient of an eluent in a single stroke syringe pump and feeds the liquid, and a liquid chromatograph including the gradient liquid feeding device.

High performance liquid chromatograph (HPLC) has become a representative means for separation and analysis of trace components. FIG. 10 shows a general HPLC configuration. The HPLC 10 sends a solvent (mobile phase) from a solvent tank 101 with a pump 102 and introduces a sample from an injector 103 into a flow path. Trace components in the sample introduced into the flow path are adsorbed and held on the column 104. Each trace component held in the column 104 is separated by gradient elution and sent to an ultraviolet spectrophotometer, a mass analyzer, or the like connected to the column 104.

The solvent flow rate in the conventional HPLC is about 0.1 to 毎 5.0 ml per minute for a normal scale HPLC, and about 1 to 100 μl per minute for a micro scale HPLC. In these normal-scale and micro-scale HPLC, a plunger pump having a reciprocating plunger and capable of infinite liquid feeding is employed as the pump 102.

In recent years, in order to separate trace components with high resolution, the flow rate of HPLC has been reduced, and a nano high-performance liquid chromatograph (nano LC) that delivers a solvent at a nanoscale flow rate has been developed. Nano LC delivers solvent at a low flow rate of about 100 to 500 nl / min. However, the above plunger pump generates a pulsating flow at the time of suction, so it is nanoscale with high accuracy and reliability. It is difficult to perform delivery at a flow rate. Therefore, in nano LC, most of the solvent delivered from the pump is discarded and only a small part is guided to the column.

FIG. 11 shows a configuration of a liquid delivery apparatus that is employed for performing gradient elution in a conventional nano LC. The gradient liquid feeding device 20 sends two kinds of solvents in the solvent tank 201 and the basket 201 to the mixer 205 by the

plunger pumps

202 and 202, respectively. The gradient liquid feeding device 20 controls the driving of the plunger pump 202 and the rod 202, thereby sequentially changing the mixing ratio of the two types of solvents and performing gradient elution on the column 104.

At this time, since the flow rate to the column 104 is set to a nanoscale of about 100 to 500 nl / min, most of the mixed solvent sent from the mixer 205 is diverted to the waste liquid tank 207 side by the flow divider 206, and only a small part. Only the flow is diverted to the column 104 side. By setting the diversion ratio in the diversion device 206 based on the permeabilities of the diversion device 206, the column 104, etc., the flow rate to the column 104 can be made nanoscale.

However, the transmittance of the flow divider 206 can change due to deterioration with time, and the transmittance of the column 104 can also change for each analysis or within the same analysis. For this reason, a variation occurs in the diversion ratio in the flow divider 206, and an unacceptable variation may occur in the flow rate to the column 104. Such variation causes a decrease in analysis accuracy.

Therefore, in the gradient liquid delivery device 20, the flow rate from the flow divider 206 to the column 104 is measured by the flow rate sensor 208, and the flow rate to the column 104 is controlled to be constant by feeding back the measured flow rate to the flow divider 206. Yes.

The sensors usable as the flow sensor 208 include a pressure measurement flow sensor and a thermal flow sensor. Among these, the pressure measurement flow sensor may not be able to compensate for the viscosity change accompanying the solvent composition change in HPLC in which the solvent composition may change during the analysis due to gradient elution. In addition, since the system volume of the pressure measurement flow sensor is usually as large as about 100 μl, it is difficult to obtain sufficient measurement sensitivity required for nano LC. For this reason, a heat flow sensor is more suitably employed.

The measurement principle of the heat flow sensor will be described with reference to FIG. The heat introduced into the channel filled with the solvent diffuses in both the upstream direction and the downstream direction by heat conduction and thermal diffusion. When one point (heating point) of the solvent is heated when the solvent in the flow path is not flowing, a temperature profile shown by a curve A in FIG. 12 appears. The shape of this temperature profile depends on the amount of heat applied to the fluid, as well as the upstream and downstream temperatures of the liquid. When the solvent in the flow path is not flowing, the solvent temperatures measured at two points (P1 and P2) that are equidistant from the heating point are equal.

On the other hand, when the solvent in the flow path is flowing, a temperature profile similar to curve B appears. That is, when the solvent is flowing, in addition to the symmetric diffusion of heat, asymmetric convection of the heated solvent occurs in the direction of the solvent flow. For this reason, when the solvent in the flow path is flowing, the solvent temperatures measured at P1 and P2 are different.

The shape of the temperature profile shown in curve B depends on the solvent flow rate (flow rate) and the resulting thermal convection. Therefore, based on the temperature difference between the solvent temperatures measured at P1 and P2, the solvent flow rate can be measured in the range of several tens of nanoliters per minute. Patent Document 1 discloses a method and apparatus for monitoring and controlling a nanoscale flow rate of a fluid in a practical flow path of an HPLC system using this heat flow sensor.

JP 2007-513338 A

In nano LC, solvent delivery is performed at a low flow rate of about 100 to 500 nl / min. However, in recent years, in order to realize further high resolution, it is required to send a solution at a very low flow rate of several nanoliters per minute. It is like that.

In the above-described conventional gradient liquid delivery device 20, the flow rate measurement can be performed with an accuracy of several tens of nanoliters per minute by adopting a thermal flow rate sensor as the flow rate sensor 208. However, when the flow dividing ratio is to be controlled in the flow divider 206 based on the measured flow rate, there is a problem that the volume of the flow divider 206 itself is too large for a flow rate of several tens of nanoliters per minute.

That is, the flow divider 206 controls the flow dividing ratio by opening and closing a valve provided inside based on the measured flow rate fed back from the flow rate sensor 208. Therefore, even if feedback of the measured flow rate is obtained with an accuracy of several tens of nanoliters per minute, the flow rate to the column 104 cannot be controlled with the same accuracy due to the system volume caused by this valve. There wasn't.

Therefore, a main object of the present invention is to provide a liquid chromatograph including a gradient liquid feeding device capable of stably and highly accurately feeding a liquid at an extremely low flow rate of several nanoliters per minute.

In order to solve the above problems, the present invention includes a gradient liquid feeding device that forms an eluent gradient in a single stroke syringe pump and feeds the liquid, and the gradient liquid feeding is provided upstream of the column for separating the components in the sample. Provided is a liquid chromatograph provided with an injection port for introducing an eluent gradient fed from an apparatus into a flow path.
In this liquid chromatograph, since a single stroke syringe pump capable of feeding a constant flow rate is employed, by appropriately setting the inner diameter of the syringe and the linear velocity of the piston, the flow rate is extremely low at several nanoliters per minute, The eluent gradient can be sent stably and with high accuracy.
This liquid chromatograph can have an eluent tank that stores an eluent mixed with a plurality of solvents at a predetermined mixing ratio and supplies the eluent to the single stroke syringe pump.
The liquid chromatograph further includes a plurality of flow paths provided with pumps for sending the solvents to the eluent tank, and control means for controlling driving of the pumps based on a set mixing ratio. It is preferable to have
In this case, the eluent tank is provided between the supply port of the solvent from the pump, a lower portion of the supply port, a discharge port for discharging the solvent to the outside, and the supply port and the discharge port. It is preferable to include a storage unit that temporarily stores the solvent flowing from the supply port toward the discharge port and supplies the solvent to the single stroke syringe pump.
Alternatively, the eluent tank is provided above the supply port of the solvent from the pump, a discharge port for discharging the solvent to the outside, and the solvent is filled between the supply port and the discharge port. And a reservoir for supplying to the single stroke syringe pump. At this time, the introduction position of the single stroke syringe pump into the storage section is opposite to the discharge port across the supply port.

In the present invention, the “single stroke syringe pump” is provided with a syringe having a syringe body capable of containing the liquid therein and a piston for sucking the liquid into the inside from the opening of the syringe body. A liquid feed pump that can suck and discharge liquid into and out of the syringe body at a predetermined flow rate without pulsating flow by moving the piston at a predetermined linear velocity based on the inner diameter of the main body. The single stroke syringe pump may be configured to store the liquid in a narrow tube (needle) attached to the opening of the syringe body, and to perform suction and discharge, in addition to the configuration of storing the liquid in the syringe body.

In the present invention, the “eluent” is a liquid used as a mobile phase of a liquid chromatograph, and means water, an organic solvent such as acetonitrile, methanol, isopropyl alcohol, or a mixed solvent thereof. To do. The eluent may include formic acid, acetic acid, trifluoroacetic acid, triethylamine, hexafluoroisopropanol (HFIP), ammonium acetate, etc. used by adding to water or an organic solvent.

According to the present invention, there is provided a liquid chromatograph provided with a gradient liquid feeding device capable of stably and highly accurately feeding a liquid at an extremely low flow rate of several nanoliters per minute.

It is a schematic diagram explaining the structure of the liquid chromatograph concerning this invention. 4 is a schematic diagram for explaining a configuration for forming an eluent gradient G in the needle 21 of the gradient liquid delivery device 2. FIG. FIG. 3 is a schematic diagram showing an embodiment of an eluent tank 25 that can be suitably employed in the gradient liquid delivery device 2. FIG. 5 is a schematic diagram showing another embodiment of an eluent tank 25 that can be suitably employed in the gradient liquid delivery device 2. 7 is a schematic diagram for explaining another configuration for forming an eluent gradient G in the needle 21 of the gradient liquid delivery device 2. FIG. FIG. 2 is a schematic diagram for explaining an operation at the time of sample feeding of an embodiment of the liquid chromatograph 1. FIG. 2 is a schematic diagram for explaining an operation at the time of eluent gradient liquid feeding according to an embodiment of the liquid chromatograph 1. FIG. 6 is a schematic diagram for explaining an operation during liquid feeding of a sample according to another embodiment of the liquid chromatograph 1. FIG. 5 is a schematic diagram for explaining an operation at the time of eluent gradient liquid feeding according to another embodiment of the liquid chromatograph 1. It is a schematic diagram which shows the structure of general HPLC. It is a schematic diagram which shows the structure of the liquid feeding apparatus employ | adopted in order to perform gradient elution in the conventional nano LC. It is a figure explaining the measurement principle of a heat flow sensor. It is a figure explaining the "delay of the rise of a gradient" which may arise with a liquid chromatograph provided with the conventional liquid sending apparatus. It is a schematic diagram explaining the structure of the liquid chromatograph using the conventional rotation valve.

Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

FIG. 1 is a schematic diagram illustrating the configuration of a liquid chromatograph according to the present invention.

The liquid chromatograph 1 includes a column 14 for separating a trace component in a sample, and a gradient liquid delivery device 2 for feeding an eluent gradient G to the column 14 to elute the trace component. . The eluent gradient G sent from the gradient liquid delivery device 2 is introduced into the flow path 145 from the injection port 15 provided upstream of the column 14 and sent to the column 14.

The gradient liquid delivery device 2 is configured by a normally used single stroke syringe pump or an improved version thereof. As shown in FIG. 1, a typical configuration of the gradient liquid delivery device 2 includes a syringe body 22 and a piston 222 for sucking liquid into the needle 21 attached to the opening 221 of the syringe body 22. It is possible to employ a configuration in which the piston 222 is moved at a predetermined linear velocity to suck and discharge liquid from the tip opening 211 of the needle 21 into and out of the needle 21. The piston 222 can be driven by rotating the screw 24 by the motor 23. The gradient liquid feeding device 2 is configured such that the entire amount of the eluent gradient G introduced into the needle 21 is discharged by pushing the piston 222 once.

As such a single stroke syringe pump, a pump capable of delivering an ultra-low flow rate of a few pl per minute is commercially available (for example, HARVARDVAPPARATUS). Also in the present invention, these commercially available syringe pumps or modifications thereof can be adopted. In the single stroke syringe pump, the needle 21 shown in FIG. 1 is not an essential component. When the needle 21 is not provided in the single stroke syringe pump, the liquid is sucked and discharged directly into and out of the syringe body 22 from the opening 221 of the syringe body 22.

The gradient liquid delivery device 2 employs a single stroke syringe pump capable of delivering a constant flow rate by rotationally driving a screw 24 by a motor 23, so that the inner diameter of the syringe body 22 and the linear velocity of the piston 222 are appropriately set. As a result, the eluent gradient G can be sent to the column 14 at a very low flow rate of several nanoliters per minute and stably and accurately.

Generally, in HPLC, an eluent gradient about 10 to 20 times its volume is sent to a column. Therefore, for example, when the volume of the column 14 is about 15 nl, liquid feeding is performed at a rate of about 5 nl / 30 minutes per minute (total amount 150 nl). In this case, the syringe body 22 preferably has an inner diameter of 50 μm and a length of about 150 mm. The inner diameter of the syringe body 22 (that is, the outer diameter of the piston 222) may be about 50 to 250 μm, and the syringe body 22 and the piston 222 can be made thinner as long as the syringe body 22 and the piston 222 can be molded.

In the gradient liquid delivery device 2, members such as the needle 21, the syringe body 22, and the piston 222 that can come into contact with the eluent gradient G are preferably made of a corrosion-resistant metal, that is, a metal or an alloy that is hardly corroded. When these members are corroded by formic acid or the like added to the eluent, metal ions are eluted from the corroded member into the eluent, causing chemical noise during analysis. In order to prevent this, the member such as the needle 21 is desirably formed of a corrosion-resistant metal, specifically, titanium, chromium, zirconia, stainless steel, gold, or the like.

Titanium, chromium, zirconia, and stainless steel oxides are very stable and hardly corroded, and form an oxide film (so-called “passive film”) on the metal surface in the air. This passive film formed by the combination of metal with oxygen is a thin and dense film, which protects the inside of the metal from acid corrosion and oxidation, and exhibits strong corrosion resistance. Titanium, etc. in the pool bear diagram (potential-pH diagram) is a “passive zone” where the progress of corrosion stops due to the formation of a passive film compared to the “corrosion zone” where corrosion progresses, and very stable and corrosive. There is a wide "dead zone (stable zone)" that is difficult to do. For this reason, the elution of metal ions into the eluent due to corrosion can be prevented by forming the member such as the needle 21 from titanium or the like. Titanium, chromium, and zirconia can be alloys with aluminum, copper, iron, manganese, molybdenum, and the like, respectively, and the ratio of chromium, nickel, and the like contained in stainless steel can be set as appropriate.

The members such as the needle 21 can be formed of gold in addition to titanium, chromium, zirconia, and stainless steel. Gold creates an immunity that is the dead zone of the pool bear diagram, and is very stable and resistant to corrosion. For this reason, by forming a member such as the needle 21 with gold, it is possible to prevent elution of metal ions into the eluent due to corrosion. In addition, as a material of the member such as the needle 21, a corrosion-resistant metal can be widely used. For example, Hastelloy (registered trademark) containing nickel as a main component and molybdenum or chromium can be used. *

FIG. 2 is a schematic diagram for explaining a configuration for forming an eluent gradient G in the needle 21 in the gradient liquid delivery device 2.

The gradient liquid feeding device 2 has an eluent tank 25 that stores an eluent in which a plurality of solvents are mixed at a predetermined mixing ratio. In the figure, the solvent A in the solvent tank a and the solvent B in the solvent tank b are mixed at a predetermined ratio by the channel 27a provided with the pump 26a and the channel 27b provided with the pump 26b, respectively, as an eluent. The case where it is supplied to the tank 25 is shown. The gradient liquid delivery device 2 drives the piston 222 in a state where the tip opening 211 of the needle 21 is positioned in the eluent supplied to the eluent tank 25, thereby making the inside of the needle 21 negative pressure. The eluent is sucked into the needle 21 (see FIG. 2A).

The

flow paths

27a and 27b are joined by a mixer 28, and the solvents A and B discharged from the

pumps

26a and 26b are mixed at a predetermined ratio, and are sent and stored in the eluent tank 25 as an eluent. Such a system is called a high-pressure gradient system because the liquids are merged and mixed at a position where pressure is applied to the liquid on the discharge side of the

pumps

26a and 26b.

In the high pressure gradient system, control means (not shown) for controlling the driving of the

pumps

26a and 26b based on the set mixing ratio is provided. In the gradient liquid delivery device 2, by this control means, for example, supply of the solvent A and B to the eluent tank 25 at a mixing ratio of 100: 0 is started, and the solvent A is gradually reduced to finally increase the solvent B. Solvents A and B can be supplied at a mixing ratio of 0: 100.

For the control means of the

pumps

26a and 26b, for example, a system in which two pumps communicate with each other or a system in which each control is performed by an external controller can be employed. Further, a binary pump having the

pumps

26a and 26b as one unit may be adopted. In any case, the mixing ratio is controlled by replacing the set mixing ratio with the driving amount (liquid feeding flow rate) of the

pumps

26a and 26b to perform liquid feeding.

In addition, in the case of carrying out mixed liquid feeding of three liquids, a solvent tank c containing solvent C, a pump 26c and a flow path 27c corresponding thereto are further provided, and the three liquids are mixed and fed by the mixer 28. A high pressure gradient system may be employed. It is possible to carry out similarly for the mixed liquid feeding of four or more liquids.

The gradient liquid feeding device 2 supplies the eluent tank 25 while changing the mixing ratio of the solvents A and B by the high-pressure gradient system composed of the

pumps

26a and 26b and the mixer 28 as described above. Then, the eluent gradient G (see FIG. 1) is formed in the needle 21 by sequentially driving the piston 222 and sucking the eluent into the needle 21.

Specifically, for example, the solvent A and B are first supplied to the eluent tank 25 at a mixing ratio of 100: 0, and a predetermined amount is sucked into the needle 21. Subsequently, after the eluent having a mixing ratio of 100: 0 stored in the eluent tank 25 is discharged from the eluent tank 25, the solvents A and B are supplied at a mixing ratio of 95: 5, and a predetermined amount is put into the needle 21. Suction. Similarly, by repeating the discharge of the eluent from the eluent tank 25, the supply of the eluent at a new mixing ratio, and the suction into the needle 21, an eluent whose concentration changes stepwise in the needle 21 can be obtained. Aspirate and form eluent gradient G.

FIG. 3 is a schematic diagram showing an embodiment of an eluent tank 25 that can be suitably employed in the gradient liquid delivery device 2. In the figure, the eluent tank 25 is indicated by a dotted line, and the eluent supplied into the tank is indicated by a solid line.

The high-pressure gradient system has a feature that bubbles are generated less because the solvent is mixed under high pressure. However, as explained in FIG. 2, in the gradient liquid feeding device 2 according to the present invention, the eluate mixed by the high pressure gradient system including the

pumps

26a, 26b, the mixer 28 and the like is eluted without applying pressure. Since the liquid is supplied to the liquid tank 25, bubbles may be generated in the eluent tank 25. In FIG. 3, the bubble is indicated by a symbol K. If the bubbles are sucked into the needle 21, the eluent gradient G cannot be formed in the needle 21 with high accuracy.

In order to prevent this, as shown in FIG. 3, the eluent tank 25 is provided with an eluent discharge port 25b below the eluent supply port 25a from the

pumps

26a, 26b, mixer 28, and the like. . Then, the eluent flowing out from the supply port 25a toward the discharge port 25b according to gravity is temporarily stored in a storage unit 25c provided between the supply port 25a and the discharge port 25b, and the needle 21 is supplied from the storage unit 25c. The eluent is supplied to the inside. In the figure, the arrow indicates the direction in which the eluent flows.

First, the eluent is supplied into the eluent tank 25 from the supply port 25a, and the eluent that has flowed out of the supply port 25a and accumulated in the storage unit 25c is sucked with the needle 21, thereby introducing the eluent into the eluent tank 25. Even if air bubbles are generated at the supply port 25a at this time, the air bubbles can be prevented from being sucked into the needle 21.

In addition, the eluent introduced into the eluent tank 25 from the supply port 25a can be discharged out of the eluent tank 25 from the discharge port 25b at any time, and the discharge of the eluent out of the eluent tank 25, The eluent gradient in which the concentration continuously changes in the needle 21 by continuously changing the solvent mixing ratio by the

pumps

26a, 26b, the mixer 28, etc. without repeating the introduction of the eluent at a new mixing ratio. G can be formed.

FIG. 4 is a schematic diagram showing another embodiment of the eluent tank 25 that can be suitably employed in the gradient liquid delivery device 2. As shown in FIG. In the figure, the eluent tank 25 is indicated by a dotted line, and the eluent supplied into the tank is indicated by a solid line.

In the eluent tank 25 shown in FIG. 4, an eluent discharge port 25b is provided above the eluent supply port 25a from the

pumps

26a, 26b, the mixer 28, and the like. The reservoir 25c between the supply port 25a and the discharge port 25b is filled with the eluent, and the eluent is supplied from the reservoir 25c into the needle 21.

At this time, the introduction position of the needle 21 into the reservoir 25c (the position of the tip opening 211 of the needle 21 introduced into the reservoir 25c) is opposite to the discharge port 25b across the supply port 25a. To. Thereby, it is possible to guide the bubbles (see symbol K) generated when the eluent is supplied into the eluent tank 25 toward the discharge port 25b and to prevent the bubbles from being sucked into the needle 21. In addition, a suction pump is connected to the discharge port 25b, and the eluent is forcibly discharged by suction to create a flow of the eluent from the supply port 25a to the discharge port 25b. Can be more effectively prevented.

The gradient liquid feeding device 2 feeds the eluent gradient G formed in the needle 21 from the injection port 15 provided upstream of the column 14 to the flow path 145 and the column 14, so that the eluent tank 25 and the injection port Drive means (not shown) capable of changing the relative position of the needle 21 with respect to 15 is provided.

The gradient liquid delivery device 2 is connected to the elution liquid suction position (position shown in FIG. 2A) where the tip opening 211 of the needle 21 is located in the eluent tank 25, and the tip opening 211 is connected to the injection port 15. The reciprocation of the eluent discharge position (the position shown in FIG. 2B) is reciprocated by being moved in the direction of arrow X in FIG. This driving means is used to immerse the tip opening 211 of the needle 21 in the eluent in the eluent tank 25 at the suction position and to crimp the tip opening 211 to the injection port 15 at the discharge position. The needle 21 is configured to be movable also in the arrow Y direction. If the needle 21 is not provided in the gradient liquid delivery device 2, the opening 221 of the syringe body 22 is immersed in the eluent in the eluent tank 25 at the suction position, and the opening 221 is connected to the injection port 15 at the discharge position. Crimp.

The gradient liquid delivery device 2 employs a single-stroke syringe pump with a constant flow rate, and aspirates and discharges the eluent by rotating the screw 24 by the motor 23, so that a minute amount of suction and discharge can be performed without causing pulsation. It can be done at a constant speed. As a result, the gradient liquid delivery device 2 can form a gradient with high precision in the needle 21 and can stably feed the formed eluent gradient, and the holding power of the column 14 can be refined. It is possible to elute a trace component while changing to.

As described above, in the liquid chromatograph 1, the gradient elution device 2 stabilizes the accurate eluent gradient and sends it at an extremely low flow rate of several nanoliters per minute, so that the subsequent absorbance detector and mass can be obtained. It is possible to obtain high measurement sensitivity and measurement accuracy in analysis using an analyzer or the like.

Further, in the liquid chromatograph 1, by adopting the gradient liquid feeding device 2, it is not necessary to perform flow rate measurement or feedback of the measured flow rate by the flow rate sensor 208 unlike the conventional gradient liquid feeding device 20 described in FIG. Therefore, it is possible to simplify the configuration of the apparatus and reduce the manufacturing cost. Furthermore, the liquid chromatograph 1 can eliminate the need for a valve opening / closing configuration similar to the flow divider 26 of the conventional gradient liquid delivery device 20, so that wear debris (impurities) generated by the valve opening / closing are mixed into the sample. It is possible to prevent a decrease in measurement sensitivity caused by doing so.

In the liquid chromatograph 1, by adopting the gradient liquid delivery device 2, it is possible to solve the so-called `` gradient rise '' problem that occurred in the liquid chromatograph equipped with the conventional gradient liquid delivery device 20. is there. In the conventional liquid chromatograph, the pumps 202 and 202 (see FIG. 11) are controlled so that the solvents A and B are supplied to the eluent tank 25 at a mixing ratio of 100: 0 as shown in FIG. 13A, for example. At the start, when the solvent A was gradually decreased and the solvent B was increased and supplied, the problem of “rising of the gradient” occurred. That is, immediately after the start of supply, a strong liquid supply pressure is applied to the mixer 205 (see FIG. 11) from the pump 202 that supplies 100% amount of the solvent A. Therefore, even if the supply of the solvent B is started from the other pump 202, it cannot be started by being pushed back by the liquid supply pressure, and the change in the concentration gradient is shown in FIG. 13B. Delays occur. This delay in rise also causes a sudden change in the concentration gradient. Therefore, accurate gradient elution cannot be performed, and an accurate analysis result cannot be obtained. In FIG. 13, the horizontal axis represents time, and the vertical axis represents the mixing ratio of solvent A and solvent B.

This delay in the rise of the gradient becomes a problem particularly when an eluent gradient is formed by the high pressure gradient system and the liquid is sent at a low flow rate. For example, in the gradient liquid delivery device 20 shown in FIG. 11, most of the mixed solvent sent from the mixer 205 by the flow divider 206 is diverted to the waste liquid tank 207, but even in this case, the flow rate to the column 104 is reduced to nanometers. In order to obtain a scale, the total flow rate of the solvent supplied from each pump 202 needs to be a low flow rate of about several tens of μl per minute. At this time, for example, if solvent A is supplied from one pump 202 at a rate of 50 μl / min, and solvent B is supplied from the other pump 202 at a rate of 1 μl / min against this liquid supply pressure, the solvent A side is pushed back. Solution B cannot be started. The effect of this pushing back increases as the total flow rate of the solvent decreases.

In contrast, in the gradient liquid feeding device 2 according to the present invention, the mixed solution of the solvents A and B is temporarily supplied to the eluent tank 25 by the high-pressure gradient system including the

pumps

26a, 26b, the mixer 28, and the like. Therefore, it is not necessary to carry out liquid feeding by the high pressure gradient system at a low flow rate. Therefore, the influence of pushing back as described above can be eliminated by increasing the total flow rate of the solvent from the

pumps

26a and 26b to, for example, about several hundred μl per minute. Therefore, in the gradient liquid feeding device 2, the mixed solvent can be fed to the eluent tank 25 in a state where there is no delay in the rising of the gradient. Then, by sequentially driving the piston 222 to suck the eluent into the needle 21 and discharging the eluent gradient G (see FIG. 1) formed in the needle 21, highly accurate gradient elution can be performed. It becomes possible.

FIG. 2 illustrates the case where the supply of the solvent A and the solvent B to the eluent tank 25 is performed by a high-pressure gradient system. The high-pressure gradient system is superior in gradient accuracy because the gradient accuracy is almost determined by the performance of each pump itself, but has a demerit in cost that a pump is required for each solvent.

On the other hand, in the conventional system called the low pressure gradient system, the gradient accuracy is inferior to that of the high pressure gradient system due to the influence of the solvent compressibility and the response of the solvent selection valve. However, there are merits such that only one liquid pump is required and the number of solvents can be easily increased. In the gradient liquid delivery device 2, a high pressure gradient system and a low pressure gradient system can be appropriately selected and adopted in consideration of these advantages and disadvantages.

When employing a low pressure gradient system, typically, a plurality of solvent tanks are connected to one pump, and a solvent selection valve is disposed between each solvent tank and the pump. The solvent selection valve can select and connect only one of the solvent tanks. The solvent selection valve sends the solvent to the eluent tank through the mixer while periodically switching the valve so that the set mixing ratio is obtained. When the low pressure gradient system is employed, the solvents are combined and mixed at the suction side of the pump, that is, at the position where the solvent is depressurized.

FIG. 5 is a schematic diagram for explaining another configuration for forming an eluent gradient G in the needle 21 in the gradient liquid delivery device 2.

The gradient liquid delivery device 2 has five eluent tanks (eluent tanks 251 to 255) that store eluents mixed in advance so that a plurality of solvents have a predetermined mixing ratio.

In the eluent tanks 251 to 255, for example, when two kinds of solvents are mixed at a predetermined ratio to form an eluent gradient, the solvents A and B are previously mixed at a mixing ratio of 100: 0 to 0: 100. Mix and store to change gradually. Specifically, for example, the eluent tank 251 is provided with solvents A and B in a mixing ratio of 100: 0, and the eluent tank 251 is provided with solvents A and B in a mixing ratio of 95: 5. The mixing ratio is changed stepwise according to the number of liquid tanks.

On the other hand, the gradient liquid delivery device 2 is provided with drive means (not shown) that can change the relative positions of the needle 21 with respect to the eluent tanks 251 to 255 and the injection port 15. The gradient liquid delivery device 2 draws the eluent in each eluent tank sequentially into the needle 21 while moving the needle 21 in the directions of arrows X and Y in FIG. A liquid gradient G is formed (see FIG. 5A). After the eluent gradient is formed, the gradient liquid delivery device 2 moves to the eluent discharge position (position shown in FIG. 5B) where the tip opening 211 is connected to the injection port 15, and the eluent gradient Is fed from the injection port 15 to the flow path 145 and the column 14.

In this way, a plurality of eluent tanks (five in FIG. 5) for storing an eluent in which a plurality of solvents are mixed in advance at a predetermined mixing ratio are provided, and the eluent gradient is drawn by sequentially sucking the eluent while moving the needle 21. Unlike the method using the high-pressure gradient system or the low-pressure gradient system described with reference to FIG. 2, the forming method does not require a pump or a control means for each pump. Can be simplified.

Next, an example of an analysis method in the liquid chromatograph 1 will be described with reference to FIGS. 6 and 7 are diagrams showing the configuration of the liquid chromatograph 1 described in FIG. 1 in more detail.

The liquid chromatograph 1 has a separation column 141 and a precolumn 142 as the column 14 (see FIG. 1). Generally, the separation column 141 and the precolumn 142 are made of silica, glass or resin beads, divinylbenzene-styrene copolymer, resin beads such as polystyrene, which are chemically bonded to alkyl groups such as methyl, butyl, octyl, octadecyl, and docosyl groups. Filled ones are used. The precolumn 142 is connected to the upstream side of the flow path of the separation column 141, and is used to temporarily hold (trap) the sample.

In the liquid chromatograph 1, the precolumn 142 is connected to the upstream of the separation column 141 by the flow path 146, and further, the eluent gradient is introduced into the precolumn 142 and the separation column 141 via the flow path 145 upstream of the precolumn 142. The injection port 15 is provided. A sample port 16 for introducing a sample into the channel is provided in the channel 146 connecting the separation column 141 and the precolumn 142. In the liquid chromatograph 1, the function of the sampler is given to the gradient liquid delivery device 2, and the sample is first fed from the sample port 16, and then the eluent gradient is fed from the injection port 15. Can be configured.

In order to cause the gradient liquid feeding device 2 to feed the sample, for example, a sample tank may be provided in addition to the eluent tank 25 shown in FIG. Then, similarly to the suction of the eluent from the eluent tank 25, the sample in the sample tank is sucked into the needle 21, and then the needle 21 is moved to a position where it is connected to the sample port 16 by the driving means described above. to eject the sample from the sample port 16 (see arrow in FIG. 6 S _1). For example, the sample tank can be provided alongside the eluent tanks 251 to 255 shown in FIG. 5, or one of the eluent tanks 251 to 255 can be used as the sample tank.

The separation column 141 has a high back pressure because a long column is packed with a finer particle size packing material than the pre-column 142 in order to obtain high resolution, and a high delivery pressure is required for the sample to pass through it. It becomes. Therefore, when a sample is injected from the gradient liquid delivery device 2 into the sample port 16, the sample introduced into the flow path 146 is fed to the precolumn 142 side without being fed to the separation column 141, and the injection port 15 through the channels head 17 and the flow channel 171 is communicated with, it is discharged to the arrow S ₂ direction. Thereby, the trace component in the sample is adsorbed to the pre-column 142.

Next, an eluent gradient is formed in the needle 21 of the gradient liquid delivery device 2 by the method described with reference to FIG. 2 or FIG. 5, and the needle 21 is moved to a position where it is connected to the injection port 15 by the driving means. The gradient is discharged (see FIG. 7).

At this time, the flow path head 17 and the flow path 171 communicated with the injection port 15 are moved relative to the injection port 15 by the driving means similarly to the gradient liquid feeding device 2, and the communication with the injection port 15 is released. Is done. At the same time, the sample port 16 is closed by the closing head 18. Similarly, the closing head 18 is configured so that the relative position with respect to the sample port 16 can be moved by the driving means. (See FIG. 6).

In the state shown in FIG. 7, when an eluent gradient is sent from the gradient liquid delivery device 2 to the injection port 15, the eluent gradient is introduced into the pre-column 142 and the trace components adsorbed in the column are eluted. At this time, since the sample port 16 is blocked by the blocking head 18, the eluted trace component is sent to the separation column 141. The minor component that is delivered to the separation column 141, after being separated and fractionated, will be sent further to the subsequent UV spectrophotometer and mass spectrometer (see arrow S _3).

6 and 7, the case where the sample is adsorbed to the pre-column 142 using the back pressure difference between the separation column 141 and the pre-column 142 when the sample is injected into the sample port 16 has been described. 8 and 9, an example of an analysis method in the liquid chromatograph 1 when there is no back pressure difference between the separation column 141 and the pre-column 142 or when it is small will be described.

In this example, a flow path head 19 is provided instead of the closing head 18 that closes the sample port 16. A separation column 141 is connected to the flow path head 19 by a flow path 191.

As shown in FIG. 8, when a sample is injected into the sample port 16 from the gradient liquid delivery device 2, the sample introduced into the flow path 146 passes through the precolumn 142, the injection port 15, the flow path head 17, and the flow path 171. Are discharged in the direction of arrow S ₂ , and trace components in the sample are adsorbed by the precolumn 142.

Next, an eluent gradient is formed in the needle 21 of the gradient liquid feeder 2, and the eluent gradient is discharged by moving the needle 21 to a position where it is connected to the injection port 15 by the driving means (see FIG. 9). At the same time, the flow path head 19 to which the separation column 141 is connected is moved by the driving means and communicated with the sample port 16. As a result, the trace component adsorbed on the pre-column 142 is eluted by the eluent gradient sent from the gradient liquid delivery device 2 to the injection port 15, and sent to the ultraviolet spectrophotometer or the like through the separation column 141. (see arrow S _3).

In this example, since the connection of the separation column 141 at the time of injection of the sample into the sample port 16 can be completely disconnected, the analysis can be performed regardless of the back pressure difference between the separation column 141 and the precolumn 142. Further, since it is not necessary to directly connect the separation column 141 and the precolumn 142, the degree of freedom of the combination of both columns can be increased.

Thus, in the analysis in the liquid chromatograph 1, the flow of the flow path for the adsorption of the sample to the pre-column 142 and the gradient elution can be switched without performing the valve switching as in the conventional liquid chromatograph. This can be done simply by moving the needle 21, the flow path head 17, the closing head 18, or the flow path head 19.

In a conventional liquid chromatograph, a rotating valve V as shown in FIG. 14 is used to adsorb a sample onto the pre-column 142 (see FIG. 14A) and gradient elution (see FIG. 14B). The flow path is switched. Specifically, in the state of FIG. 14A, the sample supplied from the arrow Q1 to the flow path passes through the rotary valve V and is discharged from the arrow Q2, and at this time, the trace component in the sample is pre-columned. Adsorbed to 142.

When the rotary valve V is rotated 60 degrees concentrically to switch the flow path (see FIG. 14B), the eluent gradient sent from the arrow R1 is introduced into the precolumn 142 this time. Thereby, the trace component adsorbed on the precolumn 142 is eluted and introduced into the separation column 141. The eluted trace components are separated and fractionated by the separation column 141, and sent out in the direction of arrow R2.

In such a conventional liquid chromatograph, the volume of the rotary valve V occupies a considerable proportion with respect to the volume of the entire flow path (system volume). In addition, there is a problem that dead volume of a sample or a sample is generated, and it takes time to feed a solvent or a sample.

The rotary valve V is usually composed of a metal stator and a resin rotor, but the stator and the rotor are closely adhered so that no leakage occurs when the rotor is driven to rotate. For this reason, metal and resin wear debris (impurities) are generated on the bonding surface between the stator and the rotor, which may be mixed into the solvent or sample. Further, in the rotating valve V, since the switching is performed in a state where the solvent and the sample are filled in the valve, the carry-over of the solvent and the sample may occur.

In conventional liquid chromatographs, due to the problems caused by the rotating valve V as described above, the analysis sensitivity and the analysis speed are reduced. In particular, in nano LC, which requires a very small amount of sample to be delivered at an extremely low flow rate, loss of sample and solvent and contamination may occur if dead volume, carryover, or contamination occurs due to valves. This causes a significant decrease in analytical sensitivity.

In contrast, in the analysis in the liquid chromatograph 1, as shown in FIGS. 6 to 9, the adsorption of the sample to the precolumn 142 and the gradient elution are performed without changing the valve, and the needle 21 of the gradient liquid delivery device 2 is used. And by moving the flow path head 17, the closing head 18, or the flow path head 19.

Therefore, there is no occurrence of dead volume, carry-over and impurities due to valve switching, and it is possible to improve analysis sensitivity by eliminating sample and solvent loss and contamination. Further, by omitting the valve, the system volume can be reduced, so that the analysis time can be shortened and the analysis efficiency can be improved.

The liquid chromatograph and the gradient liquid delivery device according to the present invention are used for separation and analysis of trace components, and can be suitably used particularly for a nano high-performance liquid chromatograph that delivers a solvent at a nanoscale flow rate. .

1 Liquid chromatograph
14 columns
141 Separation column
142 Precolumn
145, 146, 171, 27a, 27b, 27c flow path
15 Injection port
16 Sample port
17, 19 Flow path head
18 Blocking head
2 Gradient pump
21 needle
211 Tip opening
22 Syringe body
221 opening
222 piston
23 Motor
24 screws
25, 251, 252, 253, 254, 255 Eluent tank
25a Supply port
25b outlet
25c Reservoir
26a, 26b, 26c pump
28 mixer
a, b, c solvent tank
G eluent gradient

Claims

Equipped with a gradient liquid delivery device that forms an eluent gradient in the single stroke syringe pump and delivers the liquid,
A liquid chromatograph provided with an injection port for introducing an eluent gradient fed from a gradient liquid feeding device into a flow channel upstream of a column for separating components in a sample.
The liquid chromatograph according to claim 1, further comprising an eluent tank that stores an eluent mixed with a plurality of solvents at a predetermined mixing ratio and supplies the eluent to the single stroke syringe pump.
A plurality of flow paths provided with pumps for feeding each of the solvents to the eluent tank;
The liquid chromatograph according to claim 2, further comprising: a control unit that controls driving of each pump based on a set mixing ratio.
The eluent tank is
A supply port for the solvent from the pump;
A discharge port provided below the supply port for discharging the solvent to the outside;
A liquid chromatograph according to claim 3, further comprising: a storage unit provided between the supply port and the discharge port, and temporarily storing the solvent flowing from the supply port toward the discharge port and supplying the solvent to the single stroke syringe pump. Graph.
The eluent tank is
A supply port for the solvent from the pump;
A discharge port provided above the supply port for discharging the solvent to the outside;
A storage section that fills the solvent between the supply port and the discharge port and supplies the solvent to the single stroke syringe pump, and
The liquid chromatograph according to claim 3, wherein the introduction position of the single stroke syringe pump into the storage part is on the side opposite to the discharge port across the supply port.
A gradient liquid delivery system for liquid chromatographs that forms an eluent gradient in a single stroke syringe pump and delivers the liquid.