WO2009151096A1 - Liquid chromatograph and gradient liquid feed system - Google Patents
Liquid chromatograph and gradient liquid feed system Download PDFInfo
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- WO2009151096A1 WO2009151096A1 PCT/JP2009/060686 JP2009060686W WO2009151096A1 WO 2009151096 A1 WO2009151096 A1 WO 2009151096A1 JP 2009060686 W JP2009060686 W JP 2009060686W WO 2009151096 A1 WO2009151096 A1 WO 2009151096A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
- G01N2030/347—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient mixers
Definitions
- 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.
- HPLC High performance liquid chromatograph
- 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 ⁇ 5.0 ml per minute for a normal scale HPLC, and about 1 to 100 ⁇ l per minute for a micro scale HPLC.
- a plunger pump having a reciprocating plunger and capable of infinite liquid feeding is employed as the pump 102.
- Nano LC delivers solvent at a low flow rate of about 100 to 500 nl / min.
- 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.
- 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.
- the flow rate to the column 104 can be made nanoscale.
- 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.
- 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.
- 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.
- the system volume of the pressure measurement flow sensor is usually as large as about 100 ⁇ l, 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.
- 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.
- the solvent temperatures measured at two points (P1 and P2) that are equidistant from the heating point are equal.
- 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.
- solvent delivery is performed at a low flow rate of about 100 to 500 nl / min.
- it is required to send a solution at a very low flow rate of several nanoliters per minute. It is like that.
- 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.
- a thermal flow rate sensor as the flow rate sensor 208.
- 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.
- 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.
- 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.
- 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.
- a liquid chromatograph provided with an injection port for introducing an eluent gradient fed from an apparatus into a flow path.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- FIG. 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.
- 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.
- 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.
- 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.
- 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.
- the syringe body 22 preferably has an inner diameter of 50 ⁇ m 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 ⁇ m, 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.
- 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.
- a corrosion-resistant metal that is, a metal or an alloy that is hardly corroded.
- 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.
- 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.
- a corrosion-resistant metal can be widely used 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.
- 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 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.
- 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.
- control means for controlling the driving of the pumps 26a and 26b based on the set mixing ratio is provided.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the pumps 202 and 202 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.
- 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.
- FIG. 13B 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.
- the horizontal axis represents time
- the vertical axis represents the mixing ratio of solvent A and solvent B.
- 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 ⁇ l 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- the forming method does not require a pump or a control means for each pump. Can be simplified.
- 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).
- 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.
- 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.
- 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.
- 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).
- 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.
- 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).
- 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.
- the sample port 16 is closed by the closing head 18.
- 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).
- 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.
- 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).
- 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.
- 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).
- 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.
- 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.
- 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.
- the eluent gradient sent from the arrow R1 is introduced into the precolumn 142 this time.
- 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.
- 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.
- 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. .
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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
本発明は、液体クロマトグラフ及びグラディエント送液装置に関する。より詳しくは、単行程シリンジポンプ内に溶離液の勾配を形成し送液を行うグラディエント送液装置と、このグラディエント送液装置を備える液体クロマトグラフに関する。
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.
高速液体クロマトグラフ(HPLC)は微量成分の分離、分析のための代表的な手段となっている。図10に一般的なHPLCの構成を示す。このHPLC10は、溶媒タンク101からポンプ102で溶媒(移動相)を送出し、インジェクター103から流路に試料を導入する。流路へ導入された試料中の微量成分は、カラム104に吸着・保持される。カラム104に保持された各微量成分は、グラディエント溶離によって分離され、カラム104に接続された紫外線分光光度計や質量分析器等へ送液される。
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.
従来のHPLCにおける溶媒流量は、通常スケールのHPLCで毎分約0.1 ~ 5.0ml程度、マイクロスケールのHPLCでも毎分約1~100μl程度となっている。これらの通常スケール及びマイクロスケールのHPLCでは、ポンプ102として、往復動プランジャーを備え、無限送液が可能なプランジャーポンプが採用されている。
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.
近年では、微量成分を高分離能で分離するため、HPLCの低流量化が進み、ナノスケール流量で溶媒の送出を行うナノ高速液体クロマトグラフ(ナノLC)が開発されてきている。ナノLCでは、毎分100~500nl程度の低流量での溶媒送出が行われるが、上記のプランジャーポンプでは吸引の際に脈流が生じてしまうため、高い精度と信頼性を伴ってナノスケール流量での送出を行うことは難しい。そこで、ナノLCでは、ポンプから送出される溶媒の大部分を廃棄して、ごく一部のみをカラムへ誘導することが行われている。
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.
図11に、従来のナノLCにおいて、グラディエント溶離を行うために採用されている送液装置の構成を示す。このグラディエント送液装置20は、溶媒タンク201, 201中の二種の溶媒を、それぞれプランジャーポンプ202, 202によりミキサー205に送出する。グラディエント送液装置20は、プランジャーポンプ202, 202の駆動を制御することにより、二種の溶媒の混合比率を順次変化させ、カラム104でのグラディエント溶離を行う。
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.
この際、カラム104への流量を毎分100~500nl程度のナノスケールとするため、ミキサー205から送出される混合溶媒の大部分は、分流器206によって廃液タンク207側へ分流され、ごく一部のみがカラム104側へ分流される。この分流器206における分流比率を、分流器206及びカラム104等の透過率(Permeability)に基づき設定することで、カラム104への流量をナノスケールとできる。
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.
しかし、分流器206の透過率は経時劣化により変化し得るものであり、またカラム104の透過率も分析毎もしくは同一分析内で変化し得る。このため、分流器206における分流比率にばらつきが生じ、カラム104への流量に容認できないばらつきが生じることがある。このようなばらつきは分析精度の低下をもたらす。
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.
そこで、グラディエント送液装置20では、流量センサ208によって分流器206からカラム104への流量を測定し、測定された流量を分流器206にフィードバックすることによってカラム104への流量を一定に制御している。
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.
この流量センサ208として使用可能なセンサには、圧力測定流量センサや熱流量センサがある。このうち、圧力測定流量センサは、グラディエント溶離のために分析中に溶媒組成が変化し得るHPLCにおいては、溶媒の組成変化に伴う粘度変化を補償できない可能性がある。また、圧力測定流量センサは、そのシステム容積が通常100μl程度と大きいため、ナノLCに必要とされる十分な測定感度を得ることが難しい。このため、熱流量センサがより好適に採用されている。
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.
図12を参照して、熱流量センサの測定原理を説明する。溶媒が充填された流路の中に導入された熱は、熱伝導及び熱拡散によって上流方向と下流方向の両方に拡散する。流路中の溶媒が流れていない場合に、溶媒の一点(加熱点)が加熱されると、図12中曲線Aに示される温度プロファイルが現れる。この温度プロファイルの形状は、流体に加えられた熱の量、ならびにその液体の上流温度と下流温度に依存する。流路中の溶媒が流れていない場合には、加熱点から等距離に存在する2点(P1及びP2)で測定される溶媒温度は等しくなる。
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.
これに対して、流路中の溶媒が流れている場合には、曲線Bに類似した温度プロファイルが現れる。すなわち、溶媒が流れている状態では、熱の対称的な拡散に加え、加熱された溶媒の非対称的な対流が溶媒の流れの方向で生じる。このため、流路中の溶媒が流れている場合には、P1及びP2で測定される溶媒温度は異なる。
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.
曲線Bに示される温度プロファイルの形状は、溶媒の流量(流速)及びもたらされる熱対流に依存する。従って、P1及びP2で測定される溶媒温度の温度差に基づけば、溶媒の流量を毎分数十ナノリットルの範囲で測定することが可能となる。特許文献1には、この熱流量センサを用いて、HPLCシステムの実用流路内の流体のナノスケールの流量を監視し、制御するための方法および装置が開示されている。
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.
ナノLCでは、毎分100~500nl程度の低流量で溶媒送出が行われているが、近年では、さらなる高分離能を実現するため、毎分数ナノリットルという極めて低流量での送液が求められるようになっている。
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.
上述した従来のグラディエント送液装置20では、流量センサ208に熱流量センサを採用することにより、流量測定に関しては毎分数十ナノリットルの精度で行うことができる。しかし、測定された流量に基づき、分流器206において分流比率を制御しようとする際、毎分数十ナノリットルという流量に対して分流器206そのものの容積が大き過ぎるという問題があった。
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.
すなわち、分流器206は、流量センサ208からフィードバックされる測定流量に基づいて内部に設けられたバルブを開閉することにより、分流比率の制御を行っている。このため、毎分数十ナノリットルの精度での測定流量のフィードバックが得られたとしても、このバルブに起因するシステム容積のために、同等の精度でカラム104への流量を制御することはできなかった。
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.
そこで、本発明は、毎分数ナノリットルという極めて低流量の送液を安定して高精度に行うことが可能なグラディエント送液装置を備える液体クロマトグラフを提供することを主な目的とする。
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.
上記課題解決のため、本発明は、単行程シリンジポンプ内に溶離液勾配を形成し送液を行うグラディエント送液装置を備え、試料中の成分を分離するためのカラムの上流に、グラディエント送液装置から送液される溶離液勾配を流路内に導入するための注入ポートが設けられた液体クロマトグラフを提供する。
この液体クロマトグラフでは、定流量の送液が可能な単行程シリンジポンプを採用しているため、シリンジの内径及びピストンの線速度を適宜設定することで、毎分数ナノリットルという極めて低流量で、安定かつ高精度に溶離液勾配の送液を行うことができる。
この液体クロマトグラフは、複数の溶媒が所定の混合比率で混合された溶離液を貯留し、前記単行程シリンジポンプに供給する溶離液タンクを有することができる。
さらに、この液体クロマトグラフは、前記各溶媒を前記溶離液タンクに送液するためのポンプを備えた複数の流路と、設定された混合比率に基づいて各ポンプの駆動を制御する制御手段と、を有することが好適となる。
この場合、前記溶離液タンクは、前記ポンプからの前記溶媒の供給口と、供給口の下方に設けられ、溶媒を外部に排出する排出口と、供給口と排出口との間に設けられ、供給口から排出口に向かって流れる溶媒を一時的に貯留し前記単行程シリンジポンプへ供給する貯留部と、を備えることが好適となる。
あるいは、前記溶離液タンクは、前記ポンプからの前記溶媒の供給口と、供給口の上方に設けられ、溶媒を外部に排出する排出口と、供給口と排出口との間に溶媒を充満させて前記単行程シリンジポンプへ供給する貯留部と、を備えていてもよい。このとき、貯留部内への単行程シリンジポンプの導入位置は、供給口を挟んで排出口と反対側とされる。 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.
この液体クロマトグラフでは、定流量の送液が可能な単行程シリンジポンプを採用しているため、シリンジの内径及びピストンの線速度を適宜設定することで、毎分数ナノリットルという極めて低流量で、安定かつ高精度に溶離液勾配の送液を行うことができる。
この液体クロマトグラフは、複数の溶媒が所定の混合比率で混合された溶離液を貯留し、前記単行程シリンジポンプに供給する溶離液タンクを有することができる。
さらに、この液体クロマトグラフは、前記各溶媒を前記溶離液タンクに送液するためのポンプを備えた複数の流路と、設定された混合比率に基づいて各ポンプの駆動を制御する制御手段と、を有することが好適となる。
この場合、前記溶離液タンクは、前記ポンプからの前記溶媒の供給口と、供給口の下方に設けられ、溶媒を外部に排出する排出口と、供給口と排出口との間に設けられ、供給口から排出口に向かって流れる溶媒を一時的に貯留し前記単行程シリンジポンプへ供給する貯留部と、を備えることが好適となる。
あるいは、前記溶離液タンクは、前記ポンプからの前記溶媒の供給口と、供給口の上方に設けられ、溶媒を外部に排出する排出口と、供給口と排出口との間に溶媒を充満させて前記単行程シリンジポンプへ供給する貯留部と、を備えていてもよい。このとき、貯留部内への単行程シリンジポンプの導入位置は、供給口を挟んで排出口と反対側とされる。 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.
本発明において、「単行程シリンジポンプ」とは、液体を内部に収容し得るシリンジ本体と、このシリンジ本体の開口から液体を内部に吸引するためのピストンと、を有するシリンジを備えており、シリンジ本体の内径に基づいてピストンを所定の線速度で移動させることによって、シリンジ本体内外へ液体の吸引及び吐出を所定流量で脈流なく行うことが可能な送液ポンプをいうものとする。この単行程シリンジポンプは、液体をシリンジ本体内部に収容する構成の他、シリンジ本体の開口に取り付けられた細管(ニードル)内部に液体を収容し、吸引及び吐出を行う構成とすることもできる。
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.
また、本発明において、「溶離液」とは、液体クロマトグラフの移動相として用いられる液体であって、水や、アセトニトリル、メタノール、イソプロピルアルコール等の有機溶媒、これらの混合溶媒を意味するものとする。溶離液には、水や有機溶媒に添加して使用されるギ酸や酢酸、トリフルオロ酢酸、トリエチルアミン、ヘキサフルオロイソプロパノール(HFIP)、酢酸アンモニウム等が含まれ得る。
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.
本発明により、毎分数ナノリットルという極めて低流量の送液を安定して高精度に行うことが可能なグラディエント送液装置を備える液体クロマトグラフが提供される。
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.
以下、本発明を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。
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.
図1は、本発明に係る液体クロマトグラフの構成を説明する模式図である。
FIG. 1 is a schematic diagram illustrating the configuration of a liquid chromatograph according to the present invention.
この液体クロマトグラフ1は、試料中の微量成分を分離するためのカラム14と、カラム14に対し溶離液勾配Gを送液して微量成分を溶離させるためのグラディエント送液装置2を備えている。グラディエント送液装置2から送液される溶離液勾配Gは、カラム14の上流に設けられた注入ポート15から流路145内に導入され、カラム14へ送液される。
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.
グラディエント送液装置2は、通常使用される単行程シリンジポンプ又はこれを改良したものによって構成される。グラディエント送液装置2の典型的な構成としては、図1に示すように、シリンジ本体22と、このシリンジ本体22の開口221に取り付けられたニードル21内部に液体を吸引するためのピストン222とを有し、ピストン222を所定の線速度で移動させることによって、ニードル21の先端開口部211からニードル21内外への液体の吸引及び吐出を行う構成を採用できる。ピストン222の駆動は、モータ23によるネジ24の回転駆動により行うことができる。グラディエント送液装置2では、ピストン222を一回押し切ることによって、ニードル21内に導入された溶離液勾配Gの全量が吐出されるように構成されている。
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.
このような単行程シリンジポンプとしては、最小で毎分数plの超低流量送液が可能なポンプが市販されている(例えば、HARVARD APPARATUS社)。本発明においても、これら市販のシリンジポンプ又はこれを改変したものを採用できる。なお、単行程シリンジポンプにおいて、図1に示したニードル21は必須の構成とはなるものではない。単行程シリンジポンプにニードル21を設けない場合には、シリンジ本体22の開口221から直接シリンジ本体22内外へ液体の吸引及び吐出を行う。
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.
グラディエント送液装置2では、モータ23によるネジ24の回転駆動によって定流量の送液が可能な単行程シリンジポンプを採用しているため、シリンジ本体22の内径とピストン222の線速度を適宜設定することで、カラム14に対する溶離液勾配Gの送液を毎分数ナノリットルという極めて低流量で、安定かつ高精度に行うことが可能である。
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.
一般に、HPLCでは、カラムに対してその容積の10~20倍程度の溶離液勾配が送液される。従って、例えば、カラム14の容積が15nl程度である場合、毎分5nl・30分間(総量150nl)程度の送液を行う。この場合、シリンジ本体22は、内径50μm・長さ150mm程度が好適となる。シリンジ本体22の内径(すなわち、ピストン222の外径)は50~250μm程度であってよく、シリンジ本体22及びピストン222の成形が可能な限りにおいて、さらに細く形成することもできる。
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.
グラディエント送液装置2において、溶離液勾配Gと接触し得るニードル21、シリンジ本体22、ピストン222等の部材は、耐蝕性金属、すなわち腐食され難い金属又は合金によってによって形成されることが望ましい。これらの部材が溶離液に添加されたギ酸等によって腐食されると、腐食した部材から金属イオンが溶離液中に溶出して分析時の化学ノイズの原因となる。これを防止するため、ニードル21等の部材は、耐蝕性金属、具体的にはチタン、クロム、ジルコニア、ステンレス又は金等によって形成することが望ましい。
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.
チタン、クロム、ジルコニア、ステンレスの酸化物は、非常に安定で腐食され難く、空気中では金属表面に酸化物の被膜(いわゆる「不動態膜」)を形成する。金属が酸素と結びついて形成するこの不動態膜は、薄く緻密な膜であり、金属内部を酸による腐食や酸化などから保護し、強い耐蝕性を発揮する。チタン等は、プールベアダイアグラム(電位-pH図)において、腐食が進行する「腐食域」に対して、不動態膜の形成により腐食の進行が停止する「不動態域」や非常に安定で腐食され難い「不感域(安定域)」が広い。このため、チタン等によりニードル21等の部材を形成することで、腐食による溶離液中への金属イオンの溶出を防止することができる。なお、チタン、クロム、ジルコニアは、それぞれアルミニウムや銅、鉄、マンガン、モリブデンなどとの合金とすることができ、ステンレスに含まれるクロムやニッケルなどの比率も適宜設定され得るものとする。
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.
ニードル21等の部材は、チタン、クロム、ジルコニア、ステンレス以外に、金によって形成することもできる。金は、プールベアダイアグラムの不感域にあたる不感態(immunity)を作り、非常に安定で腐食され難い。このため、金によりニードル21等の部材を形成することによって、腐食による溶離液中への金属イオンの溶出を防止することができる。この他、ニードル21等の部材の材質には、耐蝕性金属を広く採用しでき、例えば、ニッケルを主成分としモリブデンやクロムを配合したハステロイ(登録商標)を採用できる。
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. *
図2は、グラディエント送液装置2において、ニードル21内に溶離液勾配Gを形成するための構成を説明する模式図である。
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.
グラディエント送液装置2は、複数の溶媒が所定混合比率で混合された溶離液を貯留する溶離液タンク25を有している。図では、溶媒タンクa内の溶媒A及び溶媒タンクb内の溶媒Bが、それぞれポンプ26aを備えた流路27a及びポンプ26bを備えた流路27bによって、所定比率で混合され溶離液として溶離液タンク25に供給される場合を示した。グラディエント送液装置2は、溶離液タンク25に供給された溶離液中にニードル21の先端開口部211を位置させた状態で、ピストン222を駆動し、ニードル21内を陰圧とすることによって、溶離液をニードル21内に吸引する(図2(A)参照)。
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).
流路27a, 27bはミキサー28で合流されており、ポンプ26a, 26bから吐出される溶媒A, Bは所定の比率で混合され、溶離液として溶離液タンク25に送液、貯留される。このようなシステムは、ポンプ26a, 26bの吐出側の液体に送圧圧力がかかる位置で各液が合流し混合されるため、高圧グラディエントシステムと呼ばれている。
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.
高圧グラディエントシステムでは、設定された混合比率に基づいてポンプ26a, 26bの駆動を制御する不図示の制御手段が設けられる。グラディエント送液装置2では、この制御手段によって、例えば、溶媒A, Bを混合比率100:0で溶離液タンク25に供給を開始し、徐々に溶媒Aを減らして溶媒Bを増やし、最終的に溶媒A, Bを混合比率0:100で供給することが可能である。
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.
ポンプ26a, 26bの制御手段には、例えば、2台のポンプが相互に通信することで制御を行う方式や、外部コントローラによってそれぞれの制御を行う方式を採用することができる。また、ポンプ26a, 26bをひとつのユニットとしたバイナリーポンプを採用してもよい。いずれの場合にも、設定された混合比率を、ポンプ26a, 26bの駆動量(送液流量)に置き換えて送液を行うことで、混合比率の制御を実現する。
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.
なお、3液の混合送液を行う場合は、さらに溶媒Cの入った溶媒タンクcと、それに対するポンプ26c及び流路27cが設けられ、ミキサー28で3液を混合し、送液する3液高圧グラディエントシステムを採用すればよい。4液以上の混合送液についても同様に行うことが可能である。
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.
グラディエント送液装置2は、このようにポンプ26a, 26b及びミキサー28等から構成される高圧グラディエントシステムにより、溶媒A,Bの混合比率を変化させながら溶離液タンク25に供給する。そして、逐次、ピストン222を駆動させて溶離液をニードル21内に吸引することによって、ニードル21内に溶離液勾配G(図1参照)を形成する。
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.
具体的には、例えば、溶離液タンク25に対し、初めに溶媒A, Bを混合比率100:0で供給し、ニードル21内に所定量を吸引する。続いて、溶離液タンク25に貯留された混合比率100:0の溶離液を溶離液タンク25から排出した後、溶媒A, Bを混合比率95:5で供給し、ニードル21内に所定量を吸引する。同様に、溶離液タンク25内の溶離液の排出と、新たな混合比率での溶離液の供給及びニードル21内への吸引を繰り返すことによって、ニードル21内に段階的に濃度変化する溶離液を吸引し、溶離液勾配Gを形成する。
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.
図3は、グラディエント送液装置2に好適に採用され得る溶離液タンク25の実施形態を示す模式図である。図は、溶離液タンク25を点線で、タンク内に供給された溶離液を実線で示している。
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.
高圧グラディエントシステムでは、高圧下で溶媒の混合を行うため、気泡の発生が少ないという特徴がある。しかし、図2で説明したように、本発明に係るグラディエント送液装置2では、ポンプ26a, 26b及びミキサー28等から構成される高圧グラディエントシステムによって混合された溶離液を、圧力がかかっていない溶離液タンク25に供給するため、溶離液タンク25内で気泡が発生してしまう可能性がある。図3中、気泡を符号Kにより示す。この気泡が、ニードル21内に吸引されてしまうと、ニードル21内で精度良く溶離液勾配Gを形成することができなくなる。
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.
これを防止するため、溶離液タンク25は、図3に示すように、ポンプ26a, 26b及びミキサー28等からの溶離液の供給口25aに対し、溶離液の排出口25bを下方に設けている。そして、供給口25aから排出口25bに向かって重力に従って流れ出る溶離液を、供給口25aと排出口25bとの中間に設けた貯留部25c内に一時的に貯留させ、この貯留部25cからニードル21内へ溶離液を供給するように構成している。なお、図中、矢印は、溶離液の流れる方向を示す。
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.
まず供給口25aから溶離液タンク25内に溶離液を供給し、供給口25aから流れ出て貯留部25cに溜まった溶離液をニードル21で吸引することにより、溶離液タンク25内へ溶離液が導入された際に供給口25aで気泡が発生したとしても、気泡がニードル21内へ吸引されるのを防止することができる。
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.
また、供給口25aから溶離液タンク25内に導入される溶離液を、随時、排出口25bから溶離液タンク25外へ排出することができ、溶離液タンク25外への溶離液の排出と、新たな混合比率での溶離液の導入を繰り返すことなく、ポンプ26a, 26b及びミキサー28等によって溶媒の混合比率を連続的に変化させることで、ニードル21内に連続的に濃度変化する溶離液勾配Gを形成することが可能となる。
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.
図4は、グラディエント送液装置2に好適に採用され得る溶離液タンク25の他の実施形態を示す模式図である。図は、溶離液タンク25を点線で、タンク内に供給された溶離液を実線で示している。
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.
図4に示す溶離液タンク25では、ポンプ26a, 26b及びミキサー28等からの溶離液の供給口25aに対し、溶離液の排出口25bを上方に設けている。そして、供給口25aと排出口25bとの間の貯留部25c内に溶離液を充満させ、この貯留部25cからニードル21内へ溶離液を供給するように構成している。
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.
この際、貯留部25c内へのニードル21の導入位置(貯留部25c内に導入されたニードル21の先端開口部211の位置)が、供給口25aを挟んで排出口25bと反対側となるようにする。これにより、溶離液タンク25内への溶離液供給時に発生した気泡(符号K参照)を排出口25bの方へ誘導し、気泡がニードル21内へ吸引されるのを防止することができる。さらに、排出口25bに吸引ポンプを接続し、吸引によって溶離液を強制的に排出して、供給口25aから排出口25bへの溶離液の流れを積極的に作り出すことにより、気泡がニードル21内へ吸引されるのを一層効果的に防止することができる。
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.
グラディエント送液装置2は、ニードル21内に形成した溶離液勾配Gを、カラム14の上流に設けられた注入ポート15から流路145及びカラム14へ送液するため、溶離液タンク25及び注入ポート15に対するニードル21の相対位置を変更し得る駆動手段(不図示)を備えている。
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.
グラディエント送液装置2は、ニードル21の先端開口部211が溶離液タンク25内に位置する溶離液の吸引ポジション(図2(A)に示す位置)と、先端開口部211が注入ポート15に接続する溶離液の吐出ポジション(図2(B)に示す位置)との2つのポジションを、上記駆動手段によって図2中矢印X方向に移動されることによって往復する。この駆動手段は、吸引ポジションにおいてニードル21の先端開口部211を溶離液タンク25内の溶離液内に浸漬させるため、また吐出ポジションにおいて先端開口部211を注入ポート15に圧着させるため、図2中矢印Y方向へもニードル21を移動可能に構成されている。なお、グラディエント送液装置2においてニードル21を設けない場合には、吸引ポジションではシリンジ本体22の開口221を溶離液タンク25内の溶離液内に浸漬し、吐出ポジションでは開口221を注入ポート15に圧着させる。
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.
グラディエント送液装置2では、定流量の単行程シリンジポンプを採用し、モータ23によるネジ24の回転駆動によって溶離液の吸引及び吐出を行うため、微量の吸引及び吐出を、脈動を生じさせることなく一定速度で行うことができる。これにより、グラディエント送液装置2では、ニードル21内に高精度に勾配を形成して、形成された溶離液勾配を安定的に維持して送液することができ、カラム14の保持力を精緻に変化させながら微量成分の溶離を行うことが可能である。
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.
以上のように、液体クロマトグラフ1では、グラディエント送液装置2によって正確な溶離液勾配を安定して、かつ毎分数ナノリットルという極めて低流量で送液することにより、後段の吸光度検出器や質量分析器等による分析において高い測定感度及び測定精度を得ることが可能となる。
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.
また、液体クロマトグラフ1では、グラディエント送液装置2を採用することにより、図11で説明した従来のグラディエント送液装置20のように流量センサ208によって流量測定や測定流量のフィードバックを行う必要がないため、装置の構成を簡略にし、製造コストの低減を図ることが可能である。さらに、液体クロマトグラフ1では、従来のグラディエント送液装置20の分流器26のようなバルブ開閉を行う構成を不要とすることができるため、バルブ開閉によって発生する磨耗カス(不純物)が試料に混入することによって生じる測定感度の低下を防止することが可能となる。
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.
液体クロマトグラフ1では、グラディエント送液装置2を採用することにより、従来のグラディエント送液装置20を備えた液体クロマトグラフで生じていた、いわゆる「グラディエントの立ち上がり」の問題を解決することも可能である。従来の液体クロマトグラフでは、ポンプ202, 202(図11参照)を制御して、例えば図13(A)に示すように、溶媒A, Bを混合比率100:0で溶離液タンク25に供給を開始し、徐々に溶媒Aを減らして溶媒Bを増やして供給しようとした場合、「グラディエントの立ち上がり」の問題が生じていた。すなわち、供給開始直後、ミキサー205(図11参照)には、100%量の溶媒Aを送液するポンプ202から強い送液圧がかかっている。そのため、溶媒Bの供給を他方のポンプ202から開始しようとしても、この送液圧によって押し返されて送液を開始することができず、濃度勾配の変化に、図13(B)に示すような遅れが生じる。この立ち上がりの遅れは、同時に濃度勾配の急激な変化も引き起こす。そのため、精度の良いグラディエント溶離を行うことができず、正確な分析結果を得ることができなかった。なお、図13中、横軸は時間、縦軸は溶媒A及び溶媒Bの混合比率を示している。
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.
このグラディエントの立ち上がりの遅れは、高圧グラディエントシステムによって溶離液勾配を形成し、低流量で送液しようとする場合、特に問題となる。例えば、図11に示すグラディエント送液装置20では、分流器206によってミキサー205から送出される混合溶媒の大部分を廃液タンク207へ分流しているが、この場合でも、カラム104への流量をナノスケールとするためには、各ポンプ202から供給される溶媒の総流量を毎分数十μl程度の低流量とする必要がある。このとき、例えば、一方のポンプ202から溶媒Aを毎分50μlで供給し、この送液圧に逆らって他方のポンプ202から溶媒Bを毎分1μlで供給しようとすると、溶媒A側の押し返しによって溶媒Bの送液を開始することができない。この押し返しの影響は、溶媒の総流量が低い程大きくなる。
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.
これに対して、本発明に係るグラディエント送液装置2では、ポンプ26a, 26b及びミキサー28等から構成される高圧グラディエントシステムにより、溶媒A,Bの混合溶液を一旦溶離液タンク25に供給する構成であるため、高圧グラディエントシステムよる送液そのものを低流量で行う必要はない。そのため、ポンプ26a, 26bからの溶媒の総流量を、例えば毎分数百μl程度にまで大きくすることで、上記のような押し返しの影響を排除することができる。従って、グラディエント送液装置2では、グラディエントの立ち上がりの遅れがない状態で、溶離液タンク25への混合溶媒の送液を行うことができる。そして、逐次、ピストン222を駆動させて溶離液をニードル21内に吸引し、ニードル21内に形成した溶離液勾配G(図1参照)を吐出させることで、高精度のグラディエント溶離を行うことが可能となる。
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.
図2では、溶離液タンク25への溶媒A及び溶媒Bの供給を、高圧グラディエントシステムによって行う場合を説明した。高圧グラディエントシステムは、各ポンプそのものの性能によってグラディエントの精度がほぼ決定するため勾配精度に優れる反面、溶媒ごとにポンプが必要となるというコスト面でのデメリットがある。
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.
一方、従来、低圧グラディエントシステムと呼ばれているシステムでは、溶媒圧縮率の影響、溶媒選択バルブの応答性などにより、勾配精度は高圧グラディエントシステムより劣る。しかし、送液ポンプが1台で済むことや溶媒数を増やしやすいなどのメリットがある。グラディエント送液装置2においては、これらのメリットとデメリットを勘案して、高圧グラディエントシステム及び低圧グラディエントシステムを適宜選択して採用することができる。
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.
低圧グラディエントシステムを採用する場合、典型的には、1つのポンプに複数の溶媒タンクが接続され、各溶媒タンクとポンプとの間に溶媒選択バルブが配設される。溶媒選択バルブは、溶媒タンクのどれかひとつのみを選択して接続することができる。この溶媒選択バルブは、設定された混合比率になるよう周期的にバルブを切り換えながら、ミキサーを介して溶離液タンクへ溶媒を送液する。低圧グラディエントシステムを採用する場合には、このようにポンプの吸引側、すなわち溶媒が減圧される位置で各溶媒が合流し混合を行う。
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.
図5は、グラディエント送液装置2において、ニードル21内に溶離液勾配Gを形成するための他の構成を説明する模式図である。
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.
このグラディエント送液装置2は、複数の溶媒が所定混合比率となるように予め混合された溶離液を貯留する溶離液タンクを5つ(溶離液タンク251~255)有している。
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.
溶離液タンク251~255には、例えば、2種の溶媒を所定比率で混合して溶離液勾配を形成する場合、溶媒A, Bを予め混合比率が100:0から0:100までの間で徐々に変化するように混合して貯留させておく。具体的には、例えば、溶離液タンク251には溶媒A, Bを混合比率100:0で、溶離液タンク251には溶媒A, Bを混合比率95:5でというように、配設する溶離液タンクの数に応じて段階的に混合比率を変化させておく。
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.
一方、グラディエント送液装置2には、ニードル21の溶離液タンク251~255及び注入ポート15に対する相対位置を変更し得る駆動手段(不図示)が設けられている。グラディエント送液装置2は、この駆動手段により、ニードル21を図5中矢印X及びY方向に移動させながら、各溶離液タンク内の溶離液を順次ニードル21内に吸引し、ニードル21内に溶離液勾配Gを形成する(図5(A)参照)。そして、溶離液勾配の形成後、グラディエント送液装置2は、先端開口部211が注入ポート15に接続する溶離液の吐出ポジション(図5(B)に示す位置)にまで移動し、溶離液勾配を注入ポート15から流路145及びカラム14へ送液する。
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.
このように複数の溶媒が予め所定混合比率で混合された溶離液を貯留する溶離液タンクを複数(図5では5つ)設け、ニードル21を移動させながら順次溶離液を吸引し溶離液勾配を形成する方式では、図2を用いて説明したような高圧グラディエントシステム又は低圧グラディエントシステムを用いた方式とは異なり、ポンプや各ポンプの制御手段が不要であるため、グラディエント送液装置2そのものの構成を簡略化することができる。
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.
次に、図6及び図7を参照しながら、液体クロマトグラフ1における分析方法の一例を説明する。図6及び図7は、図1で説明した液体クロマトグラフ1の構成をより詳しく示した図となっている。
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.
液体クロマトグラフ1は、カラム14(図1参照)として、分離カラム141とプレカラム142を有している。分離カラム141及びプレカラム142には、一般に、メチルやブチル、オクチル、オクタデシル、ドコシル基などのアルキル基を化学結合したシリカ、ガラスまたは樹脂ビーズあるいはジビニルベンゼン―スチレン共重合体、ポリスチレンなどの樹脂ビーズを充填したものが用いられる。プレカラム142は、分離カラム141の流路上流に接続され、試料を一時的に保持(トラップ)するために使用されるものである。
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.
液体クロマトグラフ1では、分離カラム141の上流に流路146によりプレカラム142が接続され、さらにプレカラム142の上流には流路145を介して溶離液勾配をプレカラム142及び分離カラム141内に導入するための注入ポート15が設けられている。また分離カラム141とプレカラム142を接続する流路146には、試料を流路内に導入するための試料ポート16が設けられている。液体クロマトグラフ1では、グラディエント送液装置2にサンプラーとしての機能を付与し、初めに試料ポート16から試料の送液を行わせ、次いで注入ポート15から溶離液勾配の送液を行わせるように構成することができる。
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.
グラディエント送液装置2に試料の送液を行わせるためには、例えば、図2で示した溶離液タンク25に試料タンクを併設すればよい。そして、溶離液タンク25内からの溶離液の吸引と同様に、試料タンク内の試料をニードル21内へ吸引させた後、上述の駆動手段によってニードル21を試料ポート16に接続する位置まで移動させ、試料を試料ポート16から吐出させる(図6中矢印S1参照)。なお、試料タンクは、例えば、図5で示した溶離液タンク251~255に併設したり、溶離液タンク251~255のうちひとつを試料用タンクに用いることもできる。
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.
分離カラム141は高分離能を得るために、プレカラム142に比べ粒子径の細かい充填剤を長いカラムに充填しているため背圧が高く、これを試料が通過するためには高い送出圧が必要となる。従って、グラディエント送液装置2から試料を試料ポート16に注入すると、流路146内へ導入された試料は、分離カラム141へ送液されることなく、プレカラム142側へ送液され、注入ポート15に連通されている流路ヘッド17及び流路171を通って、矢印S2方向へ排出される。これにより、試料中の微量成分がプレカラム142に吸着される。
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.
次に、グラディエント送液装置2のニードル21内に、図2又は図5で説明した方法によって溶離液勾配を形成し、駆動手段によってニードル21を注入ポート15に接続する位置まで移動させ、溶離液勾配を吐出させる(図7参照)。
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).
このとき、注入ポート15に連通されていた流路ヘッド17及び流路171は、グラディエント送液装置2と同様に駆動手段によって注入ポート15に対して相対移動され、注入ポート15との連通が解除される。同時に、試料ポート16は、閉塞ヘッド18によって塞がれる。閉塞ヘッド18も、同様に駆動手段によって試料ポート16に対する相対位置を移動可能に構成されており、試料ポート16に圧着されてこれを閉塞し得る位置(図7参照)と、閉塞を解除する位置(図6参照)とを往復可能とされている。
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).
図7に示す状態で、グラディエント送液装置2から注入ポート15に溶離液勾配が送液されると、溶離液勾配はプレカラム142に導入され、カラム内に吸着された微量成分が溶離される。このとき、試料ポート16は閉塞ヘッド18により塞がれているため、溶離された微量成分は分離カラム141へ送出される。そして、分離カラム141に送出された微量成分は、分離・フラクション化された後、さらに後段の紫外線分光光度計や質量分析装置へと送出されることになる(矢印S3参照)。
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).
図6及び図7では、試料ポート16への試料の注入時に、分離カラム141とプレカラム142との背圧差を利用して、プレカラム142への試料の吸着を行う場合を説明した。図8及び図9では、分離カラム141とプレカラム142との背圧差がない場合もしくは小さい場合の液体クロマトグラフ1における分析方法の一例を説明する。
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.
この例では、試料ポート16を塞ぐ閉塞ヘッド18に替えて、流路ヘッド19を設けている。この流路ヘッド19には、流路191によって分離カラム141が接続されている。
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.
図8に示すように、グラディエント送液装置2から試料を試料ポート16に注入すると、流路146内へ導入された試料は、プレカラム142、注入ポート15、流路ヘッド17及び流路171を通って矢印S2方向へ排出され、試料中の微量成分がプレカラム142に吸着される。
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.
次に、グラディエント送液装置2のニードル21内に溶離液勾配を形成し、駆動手段によってニードル21を注入ポート15に接続する位置まで移動させ、溶離液勾配を吐出させる(図9参照)。同時に、分離カラム141が接続された流路ヘッド19を駆動手段によって移動させ、試料ポート16に連通させる。これにより、プレカラム142に吸着された微量成分が、グラディエント送液装置2から注入ポート15に送液された溶離液勾配によって溶離され、分離カラム141を通って紫外線分光光度計等へと送出される(矢印S3参照)。
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).
この例では、試料ポート16への試料の注入時の分離カラム141の接続を完全に切り離すことができるため、分離カラム141とプレカラム142との背圧差にかかわらず分析が可能である。また、分離カラム141とプレカラム142を直接接続する必要がないため、両カラムの組み合わせの自由度を高めることができる。
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.
このように、液体クロマトグラフ1における分析では、プレカラム142への試料の吸着とグラディエント溶離のための流路切換を、従来の液体クロマトグラフのようなバルブ切換えを行うことなく、グラディエント送液装置2のニードル21と流路ヘッド17、閉塞ヘッド18又は流路ヘッド19を移動させることによって簡単に行うことが可能である。
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.
従来の液体クロマトグラフでは、図14に示されるような回動バルブVを用いて、プレカラム142への試料の吸着(図14(A)参照)とグラディエント溶離(図14(B)参照)のための流路切換を行っている。具体的には、図14(A)の状態では、矢印Q1から流路へ供給された試料は、回動バルブVを通過して、矢印Q2から排出され、このとき試料中の微量成分がプレカラム142に吸着される。
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.
回動バルブVを同心円上に60度回転させて流路を切換えると(図14(B)参照)、今度は矢印R1から送出される溶離液勾配がプレカラム142に導入されることとなる。これにより、プレカラム142に吸着された微量成分が溶離され、分離カラム141へと導入される。そして、溶離された各微量成分が分離カラム141により分離・フラクション化され、矢印R2方向へ送出される。
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.
このような従来の液体クロマトグラフでは、回動バルブVの容積が、流路全体の容積(システム体積)に対して、相当程度の割合を占めることとなるため、バルブ容積に起因して、溶媒や試料のデッドボリュームが発生したり、溶媒や試料の送液に時間がかかるという問題があった。
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.
また、回動バルブVは、通常、金属製のステータと樹脂製のロータとで構成されるが、ロータの回転駆動時に漏れが生じないようにステータとロータは厳密に密着されている。このため、ステータとロータの接着面において金属や樹脂の磨耗カス(不純物)が発生し、溶媒や試料に混入することがあった。さらに、回動バルブVでは、バルブ内に溶媒や試料が満たされた状態で切換えを行うため、溶媒や試料のキャリーオーバーが発生することがあった。
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.
従来の液体クロマトグラフでは、以上のような回動バルブVに起因した問題のため、分析感度や分析速度の低下が生じていた。特に、極微量の試料を極めて低流量で送液することが必要となるナノLCでは、バルブに起因してデッドボリュームやキャリーオーバー、不純物の混入が生じると、試料や溶媒のロスやコンタミネーションの原因となり、分析感度の著しい低下が引き起こされる。
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.
これに対して、液体クロマトグラフ1における分析では、図6~9に示したように、プレカラム142への試料の吸着とグラディエント溶離を、バルブ切換えを行うことなく、グラディエント送液装置2のニードル21と流路ヘッド17、閉塞ヘッド18又は流路ヘッド19を移動させることにより行うことが可能である。
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.
従って、バルブ切換えに起因したデッドボリュームやキャリーオーバー、不純物の混入の発生がなく、試料や溶媒のロスやコンタミネーションをなくして、分析感度を向上させることが可能となる。また、バルブを省略したことで、システム体積を小さくすることができるので分析時間を短縮し、分析効率を向上させることができる。
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.
本発明に係る液体クロマトグラフ及びグラディエント送液装置は、微量成分の分離・分析のために用いられ、特にナノスケール流量で溶媒の送出を行うナノ高速液体クロマトグラフに好適に採用され得るものである。
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 液体クロマトグラフ
14 カラム
141 分離カラム
142 プレカラム
145, 146, 171, 27a, 27b, 27c 流路
15 注入ポート
16 試料ポート
17, 19 流路ヘッド
18 閉塞ヘッド
2 グラディエント送液装置
21 ニードル
211 先端開口部
22 シリンジ本体
221 開口
222 ピストン
23 モータ
24 ネジ
25, 251, 252, 253, 254, 255 溶離液タンク
25a 供給口
25b 排出口
25c 貯留部
26a, 26b, 26c ポンプ
28 ミキサー
a, b, c 溶媒タンク
G 溶離液勾配
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
14 カラム
141 分離カラム
142 プレカラム
145, 146, 171, 27a, 27b, 27c 流路
15 注入ポート
16 試料ポート
17, 19 流路ヘッド
18 閉塞ヘッド
2 グラディエント送液装置
21 ニードル
211 先端開口部
22 シリンジ本体
221 開口
222 ピストン
23 モータ
24 ネジ
25, 251, 252, 253, 254, 255 溶離液タンク
25a 供給口
25b 排出口
25c 貯留部
26a, 26b, 26c ポンプ
28 ミキサー
a, b, c 溶媒タンク
G 溶離液勾配
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 (6)
- 単行程シリンジポンプ内に溶離液勾配を形成し送液を行うグラディエント送液装置を備え、
試料中の成分を分離するためのカラムの上流に、グラディエント送液装置から送液される溶離液勾配を流路内に導入するための注入ポートが設けられた液体クロマトグラフ。 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. - 複数の溶媒が所定の混合比率で混合された溶離液を貯留し、前記単行程シリンジポンプに供給する溶離液タンクを有する請求項1記載の液体クロマトグラフ。 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.
- 前記各溶媒を前記溶離液タンクに送液するためのポンプを備えた複数の流路と、
設定された混合比率に基づいて各ポンプの駆動を制御する制御手段と、を有する請求項2記載の液体クロマトグラフ。 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. - 前記溶離液タンクが、
前記ポンプからの前記溶媒の供給口と、
供給口の下方に設けられ、溶媒を外部に排出する排出口と、
供給口と排出口との間に設けられ、供給口から排出口に向かって流れる溶媒を一時的に貯留して前記単行程シリンジポンプへ供給する貯留部と、を備える請求項3記載の液体クロマトグラフ。 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. - 前記溶離液タンクが、
前記ポンプからの前記溶媒の供給口と、
供給口の上方に設けられ、溶媒を外部に排出する排出口と、
供給口と排出口との間に溶媒を充満させて前記単行程シリンジポンプへ供給する貯留部と、を備えており、
貯留部内への単行程シリンジポンプの導入位置が、供給口を挟んで排出口と反対側とされている請求項3記載の液体クロマトグラフ。 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. - 単行程シリンジポンプ内に溶離液勾配を形成し送液を行う液体クロマトグラフのグラディエント送液装置。 A gradient liquid delivery system for liquid chromatographs that forms an eluent gradient in a single stroke syringe pump and delivers the liquid.
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JP2015523540A (en) * | 2012-03-08 | 2015-08-13 | ウオーターズ・テクノロジーズ・コーポレイシヨン | Back pressure adjustment |
CN115343370A (en) * | 2021-05-13 | 2022-11-15 | 中国石油天然气股份有限公司 | System and method for quickly separating lithium element driven by micro-pressure of infusion bottle |
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JP2003014718A (en) * | 2001-06-29 | 2003-01-15 | Shimadzu Corp | Mobile-phase gradient device and high-performance liquid chromatograph using the same |
JP2003149218A (en) * | 2001-11-08 | 2003-05-21 | Nano Solution:Kk | Two-dimensional high-performance liquid chromatograph and protein analyzing apparatus using the same |
JP2006227029A (en) * | 2006-06-02 | 2006-08-31 | Hitachi High-Technologies Corp | Separation analyzer |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2015523540A (en) * | 2012-03-08 | 2015-08-13 | ウオーターズ・テクノロジーズ・コーポレイシヨン | Back pressure adjustment |
JP2014006241A (en) * | 2012-05-30 | 2014-01-16 | Arkray Inc | Liquid chromatography apparatus, liquid chromatography analysis method and liquid chromatography analysis program |
CN115343370A (en) * | 2021-05-13 | 2022-11-15 | 中国石油天然气股份有限公司 | System and method for quickly separating lithium element driven by micro-pressure of infusion bottle |
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JPWO2009151096A1 (en) | 2011-11-17 |
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