JP4643290B2 - Method and apparatus for controlling fluid with minute flow rate - Google Patents

Description

The present invention relates to a method and apparatus for controlling a fluid with a minute flow rate.

  Means for branching the flowing fluid, such as introducing a part of the fluid into another device, obtaining a flow rate according to the purpose, or collecting a part of the fluid separately, is important.

As a method for dividing the fluid, for example, as shown in FIG. 1, a resistor is incorporated in a line, and a method of obtaining a target flow rate by the pressure of the resistor is used.
As shown in FIG. 1, the fluid 1 flowed from the inlet at a flow rate Q1 is branched and introduced into the resistors 4, 5, and 6 having different pressures. The flow velocity Q1 is the sum of the 2-side flow velocity Q2 and the 3-side flow velocity Q3, and the pressures on the 2-side and 3-side are equal and the following equation is established.
Q1 = Q2 + Q3 r1 × u × Q2 + r2 × u × Q2 = r3 × u × Q3
r1: Resistance coefficient of the resistor R1, r2: Resistance coefficient of the resistor 6, r3: Resistance coefficient of the resistor 4, u: Fluid viscosity Two-side flow velocity Q2 = r3 / (r1 + r2 + r3) Q3. That is, a required flow rate can be obtained depending on the resistor used.

In particular, it is an indispensable technique in the analytical field dealing with trace components, and is used for branch introduction into different apparatuses such as mass spectrometers, purification for collecting trace amounts of liquid, and HPLC analysis.
For example, in LC-MS analysis often used for biological sample analysis, it is necessary to send liquid to MS at a flow rate of 4 μL / min or less, but there are few general-purpose LC pumps that can flow well in that region. For this reason, there are methods in which the mobile phase is branched before entering the analysis column with pressure resistance, or the mobile phase that has come out of the analysis column is branched to flow the flow rate necessary for MS. This analyzer has been commercialized as a device that adjusts the split ratio using a pressure regulator, and a device that attaches a thin capillary tube and splits it with its resistance ratio. It is used by connecting between pump columns and column MSs. It has been broken.
In these apparatuses, the flow rate obtained is limited to 2 μL / min, and accurate branching at a low flow rate cannot be performed.

However, the target sample actually required is valuable, and it is necessary to analyze a very small amount of sample with high sensitivity. As the HPLC column, a capillary nano HPLC column having an inner diameter of 0.2 mm or less is used, and a flow rate of 2 μL / min or less is required. Furthermore, since the detection sensitivity in a mass spectrometer increases as the flow rate decreases, a flow rate of 0.5 μL / min or less is required. However, it cannot be used with the current splitter, which has the limit of obtaining a flow rate of 2 μL / min.
The cause is that there is no resistor that can obtain a stable load at a low flow rate for accurate branching.

As one embodiment of the splitter, there is also a method in which the low flow rate is obtained by simply splitting at a ratio of the column pressure to the resistance tube as shown in FIG. In this structure, when the liquid sent from the pump reaches the branching portion, it is branched according to the pressure resistance (split ratio) between the analytical column and the waste liquid side resistor, and sent to the column.
For example, in the HPLC system as shown in FIG. 2, if a resistor having a diameter of 30 μm FS silica tube having a length of 20 cm and a pressure of 5 Mpa at a pump flow rate of 40 μL / min is used as the resistor, the flow rate is 0.4 μL / min. A mobile phase can be branched and introduced into an analytical column of 5 Mpa in min. However, the split ratio is large, and the reproducibility of the flow rate flowing to the column side cannot be obtained due to the influence of outside air. In addition, most of the eluent used for actual analysis is discarded, which is wasted.
In order to obtain a reproducible flow rate, a split of 1/10 or less is ideal, but when trying to obtain such a split ratio with a hollow pipe such as an FS silica tube, a 30 μm FS tube with a narrow inner diameter is used. Even if it is, it is necessary to attach a resistance tube of 1 m or more.
If it is longer, it becomes clogged and cannot be practically used. In such a small-diameter tube, several tens of centimeters is the limit, and a split of 1/10 or less cannot be obtained.
Actually, when a liquid flow was sent to a resistance tube using a fused silica tube having an inner diameter of 0.03 mm and a length of 500 cm at a flow rate of 1 μL / min, the liquid supply pressure at the start of liquid supply was 4.8 MPa Thus, after 2 hours of liquid feeding, the pressure rises to 10 MPa, and after 2.5 hours, the liquid feeding pressure rises to 35 MPa or more. The liquid could not be sent.

In addition, instead of a resistance tube, a packed column for HPLC packed with particles that can obtain a split ratio of about 1/10 can be used. However, particle packing causes packing reproducibility and changes in packing due to pressure fluctuations. It is easy to obtain a stable pressure.
Furthermore, a column with the desired resistance cannot be made freely. Naturally, a filling work is required, and the resistor is expensive.
Furthermore, since the flow velocity is naturally faster on the resistance tube side, clogging is likely to occur.
When the branched components are introduced into the MS or when the branched components are recovered, the filler particles may be adsorbed to change the components contained in the liquid.
The following various methods have been proposed as a method for manufacturing a monolith structure.
JP-A-6-265534, JP-A-7-247180, JP-A-7-501140, JP-A-6-107461.

JP-A-6-265534 JP 7-247180 A JP 7-501140 A JP-A-6-107461

In order to obtain the desired flow rate, especially in the analytical field where it is necessary to deal with trace components during the flow of liquid or gas, stable resistance without variation is obtained even at low flow rates, and component changes Equipment that does not occur is indispensable.
In order to obtain a low flow rate by splitting the column pressure and resistance tube ratio, the tube with a narrow inner diameter must be formed thin and long as the resistance tube, which makes it easy to clog and cause pressure fluctuations. There is a demand for a resistor that can be eliminated, formed short, and has little pressure fluctuation.
In the present invention, by using a monolith structure that can form a desired through-pore by selecting a starting material at the time of production, resistance according to the purpose is obtained, and there is no change in space even under high pressure, and stable. In addition to the tube, it can be used not only in the tube but also in various shapes such as a filter, can be incorporated into various devices in various shapes, and can be adsorbed by selecting starting materials. We are proposing a resistor that can control its performance and devices and instruments using it.

In order to achieve the above-mentioned object, first, a monolith structure having a continuous hole of 0.5 μm to 100 μm penetrating from the inlet to the outlet is used as a resistor, and the flow of liquid or gas is branched to form a monolith structure. A fluid control method with a minute flow rate to obtain a desired flow velocity corresponding to a body. 2ndly, the mesopores of a monolith structure are less than 2 nm, The fluid control method of the micro flow rate of Claim 1 characterized by the above-mentioned. Third, a device for obtaining a desired flow velocity by branching the flow of liquid or gas , and 0.5 μm to the inside of the flow path after branching at the branching part or branching tool of the device from the inlet to the outlet A fluid control device comprising a resistor composed of a monolith structure having continuous through holes of 100 μm. Fourth, the fluid control device according to claim 3 , wherein mesopores of the monolith structure are less than 2 nm. Fifth, the fluid control device according to claim 3 or 4, wherein the resistor is created in a flow path having an outer diameter of 0.2 mm or less connected to an analytical instrument such as a mass spectrometer. suggest.

According to the invention of claim 1, by using a monolith structure having a continuous hole of 0.5 μm to 100 μm penetrating from the inlet to the outlet as a resistor, the flow of liquid or gas is branched to form a monolith structure. Since a corresponding desired flow rate is obtained, resistance to a stable fluid without variation can be obtained even at a low flow rate by using a monolith structure that can form a desired sulpore. The desired target flow rate can be obtained without fluctuation of the space even under high pressure.
As a result, it is possible to provide a method capable of performing accurate branching at a flow rate of 2 μL / min or less necessary for mass spectrometry or HPLC analysis, which is required to analyze a very small amount of sample with high sensitivity.
According to the invention of claim 2, since the mesopores of the monolith structure are less than 2 nm, in addition to the effect of the invention of claim 1, the pressure becomes constant and the time until stabilization becomes short, so that the dust Therefore, the flow of the sample is not affected under high pressure, so that the flow can be quickly and stably branched at a lower flow rate, and a higher sensitivity analysis is possible.

According to a third aspect of the present invention, there is provided a device for obtaining a desired flow velocity by branching the flow of liquid or gas , wherein the inlet into the flow path after branching by the branching part or branching tool of the device is provided. Since a resistor composed of a monolith structure having a continuous hole penetrating from 0.5 to 100 μm from the outlet to the outlet is installed, 30 to 80% of the skeleton for forming the monolith structure exists, compared to a pipe of the same diameter, It can be formed as an internal volume of 20 to 70%, has a small delay time, and is extremely excellent as a resistor. However, a stable resistance with no pressure fluctuation can be obtained due to its structure, and a stable resistance without variation can be obtained even at a low flow rate at which no component change occurs. In addition, the monolith structure of the present invention provides a resistance inversely proportional to the square of the sulpore, and can provide a high pressure even at a low flow rate.
Accordingly, it is possible to provide a fluid control apparatus in which the length can be formed short, and a short resistor that can be branched in a short time and has no clogging is installed . Further, since the pressure is inversely proportional to the cross-sectional area of the tube to be created, the resistance can be freely controlled in accordance with the control of the sulpore according to the purpose.

Further, according to the fourth aspect of the present invention, since the mesopores of the monolithic structure is less than 2 nm, in addition to the effect of claim 3, improved the instability due to flow variations, the flow rate is reproducible It can be obtained quickly and stably.

Furthermore, according to the invention of claim 5, since the resistor is created in a flow path having an outer diameter of 0.2 mm or less connected to an analytical instrument such as a mass spectrometer, uniform droplets can be formed, Without connecting a sprayer, it was possible to introduce a directly branched liquid and to easily ionize the droplets.
  As a result, the apparatus itself is simplified, the ionization apparatus is also inexpensive, the ionization sensitivity is improved, and the spray ionization method is made more effective.

Various methods as described above can be used as a method for producing a monolith structure used in the present invention.
  It is made of glass or glass ceramics, has a through-hole having a pore diameter of 500 nm or more and continuous on a three-dimensional network, and a pore having a pore diameter of 5 to 100 nm formed on the inner wall surface of the through-hole, and the total volume of the pore is Inorganic porous, characterized in that it is 10 m3 / t or less, the volume ratio occupied by through-holes is 20 to 90%, and the volume ratio for closing pores in all pores is 10% or more column. (See JP-A-6-265534) An organometallic compound containing a non-hydrolyzable organic functional group bonded via at least one metal / carbon bond and a hydrolyzable functional group is hydrolyzed. After polymerization and gelation of the reaction solution system, unnecessary substances in the gel are removed and heated. Gelation of the reaction solution system by hydrolyzing and polymerizing an organometallic compound containing a non-hydrolyzable organic functional group bonded via at least one metal / carbon bond and a hydrolyzable functional group A method for producing an inorganic porous material to which an organic functional group is bonded, wherein an unnecessary substance in the gel is removed after heating. (See JP-A-5-501935)

  By adding a degassed polymerization mixture containing porgen to a tube closed at both ends, polymerizing the mixture to produce a macroporous organic polymer flag and washing the macroporous polymer graph. A liquid chromatography column made in a manner that removes the porogen and produces a plug containing small pores with a diameter of about 200 nm ends and large pores with a diameter of about 600 nm or more. Gelation of the reaction solution system by hydrolyzing and polymerizing an organometallic compound containing a non-hydrolyzable organic functional group bonded via at least one metal / carbon bond and a hydrolyzable functional group A method for producing an inorganic porous material to which an organic functional group is bonded, wherein an unnecessary substance in the gel is removed after heating. (A) A solution of titanium alkoxide represented by the formula: Ti (OR) 4 in which R is alkyl having 1 to 6 carbon atoms is prepared, and (b) water is added to each of the obtained alcoholcosides. A stoichiometric amount equal to one alkoxide group of the molecule to all alkoxide groups of each alkoxide molecule are mixed in an equal stoichiometric amount and heated at a given temperature for a given time to cause hydrolysis and condensation. (C) The moldable sol obtained in step (b) is formed into a film or monolith, and then (d) the formed film or monolith is heated to a predetermined temperature at a rate of 1000 ° C. or higher. The layer is cured by holding for 1 second to 60 minutes, and then cooled to room temperature.

Since it is used under high pressure, a monolith body or an inorganic porous body containing an organometallic element having a higher withstand pressure than a polymer resin monolith structure is suitable for the resistance tube.
In particular, a monolith body using an organometallic compound containing silicon element or titania element as a starting material is inactive and is suitable as a resistor because of its high breakdown voltage.
In addition, in the monolith structure, mesopores may exist in addition to the spore pores through which liquid flows in the production process, but the presence of these mesopores takes time until the pressure becomes constant, For mesopores, a monolith structure of less than 2 nm that does not affect under high pressure is more suitable.
Since the mesopores can be immersed in less than 2 nm by firing at 900 ° C. or more, a monolith body having mesopores may be fired.
Considering the comparison between a resistor made of such a monolith structure in a pipe and a resistance pipe of a conventional thin pipe, the monolith structure has a continuous through-hole on a three-dimensional network, and the through-hole is a conventional one. This corresponds to the inner diameter of a thin pipe.
For example, an equivalent resistance can be obtained with a monolith structure with a sulpore of 30 μm that is the inner diameter of the through hole and a pipe with an inner diameter of 30 μm. In the pipe portion having an inner diameter of 30 μm, fluid flows in the pipe portion, and clogging due to dust or the like occurs, so that it cannot be used as a resistor. However, in the monolith structure, the through holes are connected to each other in a three-dimensional manner, The clogging is influenced by the diameter of the entire monolith body, in this case, the inner diameter of the created pipe. By using a monolith structure, a resistor without clogging can be obtained. The skeleton for forming the monolith structure is present in the pipe in an amount of 30 to 80%, and the internal volume can be 20 to 70% as compared with a pipe having the same diameter. A smaller internal volume is suitable as a resistor because a delay time does not occur. Here, the resistor may refer to the monolith structure itself or may be formed in various shapes such as a tube and a pipe.

The monolith structure can be freely created by using a catalyst that coexists with the initial monomer species and the amount of monomer, etc., but the monolith structure is prepared by using an organic silane that can withstand pressure and does not cause adsorption. I investigated.
The formamide ratio affecting sulpore was changed, methyltrimethoxysilane, formamide as a coexisting substance and 1 mol / l nitric acid aqueous solution as a catalyst were mixed at a ratio of 1: 2.5: 2.8 to obtain a uniform solution. . Sulpore 1 μm (Table 1 resistors 2A to 4A)
Put into a fused silica column with an inner diameter of 0.1 mm and a length of 100 mm under nitrogen gas pressure, seal both ends with a polypropylene seal film, gel at 15 ° C. for 15 hours, and then wash with 2 mL of methanol to obtain a monolith structure Eight resistors 2A made of
The same solution was simultaneously put into a fused silica column having an inner diameter of 0.05 mm and a length of 5 m, both ends were sealed with a polypropylene sealing film, gelled at 40 ° C. for 15 hours, washed with 2 mL of methanol, and then from the monolith structure. A resistor was obtained.
The resistor was cut to a length of 70 mm to obtain 7 resistors 3A and a length of 170 mm to obtain 7 resistors 4A.

The ratio of formaldehyde that affects sulpores is changed, and methyltrimethoxysilane, formaldehyde as a coexisting substance, and 1 mol / 1 nitric acid aqueous solution as a catalyst are mixed in a molar ratio of 1: 1.8: 2.8 to obtain a uniform solution. Got.
Put into a fused silica column with an inner diameter of 0.1 mm and a length of 150 mm under nitrogen gas pressure, seal both ends with a polypropylene sealing film, gel at 15 ° C. for 15 hours, and then wash with 2 mL of methanol to obtain a monolith structure Eight resistors 1A consisting of:
When one piece and each piece were observed with a scanning electron microscope (SEM), a gel skeleton and through-holes continuous in a three-dimensional network were observed. Further, when the monolith rod prepared from the residual solution was inspected by the nitrogen adsorption method, it was observed that the mesopores were less than 2 nm.

モノリス構造体からなる抵抗体は、スルポアの２乗に反比例した抵抗が得られ、低流量でも高圧が得られることが分かった。又、圧力は、作製する管の断面積にも反比例するので、目的に応じた抵抗をスルポアの制御と合せて自由に作られることになる。 An SEM photograph of the resistor 1A is shown in FIG. It was found that the diameter of the through hole, which is a through hole, was 2 μm. The SEM photograph of the resistor 2A and the SEM photograph of the resistor 3A are shown in FIGS. As a result, it was confirmed that the monolith structures made from the same proportion of solution were not affected by the inner diameter and were also 1 μm sulpores.
Ion exchange water was allowed to flow through these resistors, and pressure reproducibility was examined.
The results are shown in Table 1.

It has been found that a resistor made of a monolith structure has a resistance inversely proportional to the square of the sulpore, and a high pressure can be obtained even at a low flow rate. Further, since the pressure is inversely proportional to the cross-sectional area of the tube to be produced, the resistance according to the purpose can be freely created in combination with the control of the sulpore.

For comparison, when a flow rate of 1 μL / min was passed through an FS silica tube having an inner diameter of 0.03 mm and a length of 500 cm, a pressure of 4.8 Mpa close to that of the resistor 1A was obtained, but after 2 hours, it exceeded 10 Mpa, After 2.5 hours, it exceeded 35 MPa and could not flow. This was because clogging due to debris such as pump seals occurred because the flow path was long.
Further, as a comparison, throughpores 2 [mu] m, the four lots of HPLC monolithic capillary columns are commercially available mesopores 10nm (MonoCap ^R C18), were investigated pressure resistance was fed at 1 [mu] L / min. The time from the beginning of flow until the pressure was stabilized took 10 times or more that of the resistor of the present invention having small mesopores, and stabilized after 10 minutes or more had elapsed. It is considered that it took time for the liquid to enter and exit the mesopores, and that it took much longer to stabilize than the resistor of the present invention having small mesopores. As a resistor for performing branching, it takes a long time to stabilize, which causes a problem because the branched flow does not follow the inlet flow. When used at the inlet of a column such as HPLC, it takes time for the column to equilibrate and there is no significant effect. However, after separation, when branching directly to each device, or when introduced into a mass spectrometer, it becomes a fatal defect and cannot be used as a resistor for branching.
An important condition for the resistor is that the mesopores are less than 2 nm.

Moreover, the pressure 1 hour after the start of liquid feeding was 3.5 Mpa, 4.5 Mpa, and 4.8 Mpa depending on the lot. The cause is considered to be a variation of the monolith structure due to the formation of mesopores, but it is not suitable as a resistor because it cannot be controlled stably.
Furthermore, the existing commercially available monoliths have chemical species bound thereto, and it is considered that the fluid is contaminated by dropping of the bound chemical species. Moreover, it is naturally expensive and is not actually used as a resistor.
A monolith structure having a mesopore of less than 2 nm is effective as a resistor because a sufficient pressure can be obtained as a resistor even at a low flow rate and the resistance can be freely controlled.

In the apparatus shown in FIG. 2, when the resistor 2A having the sulpore μ1 m of Example 1 is cut into 2 cm and used as the waste-side resistor 12, a column capable of obtaining a pressure of 4.8 Mpa when fed at 1 μL / min as the column 11 (Fig. 6), 80% methanol eluent was sent from the pump at a flow rate of 3 μL / min, and the elution time of amylbenzene was examined. As a result, high reproducibility was obtained from 9.8 to 9.95 minutes. It was.
In addition, when an HPLC column capable of obtaining a pressure of 3.5 Mpa when a liquid was fed at 1 μL / min in the same system, the same high reproducibility as 8.76 to 8.87 minutes was obtained. It was. (Fig. 7)
The difference in elution time between the above two examples was that the split ratio changed due to the difference in the liquid feeding resistance of the column (4.8 Mpa and 3.5 Mpa), and the elution time was earlier at 3.5 Mpa. (Fig. 7)
If a resistor having a monolith structure with a sulpore of 0.5 to 20 μm is used in the tube, a shorter resistance tube can be formed at a high pressure, and a monolith structure can be directly formed in the resistance tube, so there is no filling process. Homogeneous and high reproducibility was obtained.
Column: Mono Cap Fast-flow 100μm × 270mm
Eluent: methanol / water = 80/20
Flow rate: Nanosplitter is always 3.0 μL / min. Column temperature: Room temperature (20 ° C. to 25 ° C.)
Sample 1. 1. Uracil Toluene3. Ethylbenzene4. Propylbenzene
5. Butylbenzene Amylbenzene

In the splitting method shown in Example 2, the pressure fluctuation of the column directly changes the split ratio, and the flow rate sent to the analytical column greatly changes due to the change of the split ratio. Variations in the flow rate in the analysis are directly related to the analysis result, and the analysis may be impossible. Analytical column pressure fluctuations that can cause this phenomenon are due to differences between lots due to differences in packing conditions in packed analytical columns, fluctuations due to changes in packing conditions due to use, and column wear and deterioration. It is a phenomenon that can easily occur.
As a method for reducing the influence of such column pressure fluctuation, the configuration shown in FIG. 8 in which the internal resistor 13 is also inserted in front of the column is ideal. The shorter the internal resistor used for this, the faster the liquid replacement, and the leakage of particles causes clogging of the column. Therefore, a high pressure can be obtained even when the internal resistor is short, and there is no leakage of particles. The use of is inevitable.

  In the configuration of FIG. 8, the liquid sent from the pump 9 is branched into two flow paths by a branching tool, one branched to the column side and the other to the waste liquid side. A column side resistor 13 (internal resistance) and a waste liquid side resistor 12 are arranged in each flow path, and an analysis column is connected downstream of the column side resistor. At this time, the design is made so that the liquid feeding resistance of the column side resistor 13 is sufficiently larger than the liquid feeding resistance of the column. By doing this, even when the liquid feeding resistance of the column changes, the total fluctuation of the resistance on the column side after the branching tool becomes very small, so the solution branching is the column side resistor (internal resistance) And liquid feeding according to the split ratio generated by the resistance ratio of the waste liquid side resistor is ensured.

Thus, when a resistor is arranged on the upstream side of the column, a resistor using a long fused silica tube as in the prior art has a large dead volume until the liquid sent from the pump reaches the column. End up. For this reason, particularly when gradient analysis is performed, the dead volume becomes a delay time until the gradient starts, resulting in a long time for the analysis. One way to solve this is to use a packed column as the resistor. In this case, there is a possibility that the packing state will change due to the liquid feeding, or the downstream of the resistor due to dropout of particles. May cause clogging of connected analytical columns.
On the other hand, when the monolith resistor of the present invention is used, a high liquid feeding resistance can be obtained with a short resistor, so that the dead volume in the resistor can be suppressed.

In the configuration of FIG. 8, a monolith resistor 12 having an inner diameter of 0.1 mm and a length of 7 cm and a sulpore of 1 μm are used on the waste liquid side, and a monolith resistor 13 having an inner diameter of 0.05 mm and a length of 10 cm and a sulpore of 1 μm are used on the column side. Then, an 80% methanol eluent was fed from the pump 9 at a flow rate of 3 μL / min, and the retention time of amylbenzene was measured.
A high reproducibility of 9.8 to 10.0 minutes, an average of 9.9 minutes, and a deviation value of 0.4% was obtained on a column of 2.7 to 5.4 Mpa. (Table 2)

As the optimum embodiment shown in the third embodiment, the forms shown in FIGS.
In the configuration of FIG. 9, the flow path from the pump is connected via the connector 14 and fed into the splitter device 15. The sent liquid passes through the flow path of the raw tube 16 and reaches the dispensing device 18. The liquid that has reached the branching portion 18 is branched depending on the ratio of the liquid feeding resistance of the waste liquid side monolith resistor 17 and the column side monolith resistor 19 that follows. The liquid passing through the column side monolithic resistor 19 is connected to the column via the splitter outlet side connector 20. The connector 14 for connecting the flow path from the pump has a built-in filter, and can prevent intrusion of dust.

The configuration of FIG. 10 is an apparatus diagram in which two types of column-side resistors 21 and 22 are installed.
As the resistors to be used, the monolithic resistor 7 cm of the present invention having an inner diameter of 50 μm as the column-side resistor 21, the monolithic resistor 17 cm of the present invention having an inner diameter of 50 μm as the column-side resistor 22, and the inner diameter of 100 μm as the waste-side resistor 17. 9 cm of the present monolith-filled resistor was used. The liquid supply pressure when a 20% acetonitrile solution was supplied to each resistance tube at a flow rate of 0.4 μL / min was 15 Mpa for the column side resistor 21, 36 Mpa for the column side resistor 22, and 4 Mpa for the waste liquid side resistor 17. It was. The sulpore of the monolith structure used in the embodiment of FIGS. 9 and 10 is 1 μm.

  As selection criteria for each resistor, the split ratio was calculated based on the optimum flow rate of the nanocolumn to be used (inner diameter: 100 μm) and the optimum flow rate (2 to 3 μL / min) of a commonly used micropump. In addition, the resistor upstream of the column is sufficiently larger than the resistance of the column, and the liquid feeding resistance from the pump needs to be appropriate in order to reduce the load on the pump. A higher resistance body is not subject to fluctuations in the pressure of the column, but liquid supply at a high pressure from the pump is required as the resistance increases. It is desirable to avoid liquid feeding under high pressure as much as possible in order to accelerate the deterioration at the same time as there is an allowable range of the pump itself.

内径１００μｍのカラムは、抵抗体２１に接続し、ポンプの流速を３μＬ／ｍｉｎとすることにより、０．５μＬ／ｍｉｎのカラム流速が得られ、内径７５μｍのカラムは、抵抗体２１に接続し、ポンプの流速を２μＬ／ｍｉｎとすることにより、０．３μＬ／ｍｉｎのカラム流速が得られ、内径５０μｍのカラムは、抵抗体２２に接続し、ポンプの流速を２μＬ／ｍｉｎとすることにより、０．１μＬ／ｍｉｎのカラム流速が得られる設計とした。
実際に上記の設定スプリッター１５を用いて表３に示した各内径の分析カラムを測定した結果、表４のような結果が得られた。異なる種類の夫々のカラムで本発明モノリス抵抗体を用いた分岐では、最適の流速が得られた。

実際の使用に際しては、使用しないカラム側出口にプラグを設置し、使用しない抵抗体への流入を遮断するか、もしくは使用しないカラム側出口に充分大きな抵抗体を接続することにより、使用しない抵抗管への液体の流入を充分無視できる状態にする。 The optimum flow rate of a general column is 0.5 μL / min for a column with an inner diameter of 100 μm, 0.3 μL / min for a column with an inner diameter of 75 μm, and 0.1 μL / min for a column with an inner diameter of 50 μm. (Table 3) However, the pressure varies depending on the monolith and the particle packing type.

The column with an inner diameter of 100 μm is connected to the resistor 21, and the column flow rate of 0.5 μL / min is obtained by setting the flow rate of the pump to 3 μL / min. The column with an inner diameter of 75 μm is connected to the resistor 21, By setting the flow rate of the pump to 2 μL / min, a column flow rate of 0.3 μL / min is obtained, and the column having an inner diameter of 50 μm is connected to the resistor 22 and the flow rate of the pump is set to 2 μL / min. Designed to obtain a column flow rate of 1 μL / min.
As a result of actually measuring the analytical columns having the respective inner diameters shown in Table 3 by using the setting splitter 15, the results shown in Table 4 were obtained. In the branch using the monolith resistor of the present invention in each of the different types of columns, the optimum flow rate was obtained.

In actual use, install a plug at the unused column-side outlet to block the flow into the unused resistor, or connect a sufficiently large resistor to the unused column-side outlet. The flow of liquid into the tank is in a state where it can be ignored sufficiently.

In the above combination, the gradient delay was evaluated (FIG. 11).
A pump (MP711) and a UV detector (MU701) were connected to the splitter, and the change in absorbance was monitored under the following conditions. The monitoring results are as shown in FIG.
Analysis conditions A. MeOH; B, 0.5% acetone in MeOH
A / B
90 / 10- (10 min) -90 / 10-80 / 20- (10 min) -80 / 20-70 / 30- (10 min) 70 / 30-60 / 40- (10 min) -60 / 40-50 / 50 -(10min) -50 / 50-40 / 60- (10min) -40 / 60-90 / 10
Detection wavelength: 254 nm
Table 5 shows the delay times. In the table, the used channel Column 1 is the result of using 21 in FIG. 10 and the Column 2 is the result of using 22.

図１０に示したスプリッター１５を用いて、グラジェントの再現性を評価した。
ナノフロースプリッターにポンプ（ＭＰ７１１）とＵＶ検出器（ＭＵ７０１）を接続し、評価には抵抗体２２側の接続部を使用した。評価時のカラム側の流速は０．１６μＬ／ｍｉｎであった。図１２が示すように、０．１６Ｌ／ｍｉｎという微少流量域でも高い再現性（Ｎ＝３）があることが分かった。
これは、モノリス抵抗体が安定した抵抗を生ずるものであることを証明している。
分析条件
Ａ．ＭｅＯＨ；Ｂ，０．５％acetone （in MeOH）
Ａ／Ｂ：９０／１０−（５ｍｉｎ）−９０／１０−（１５ｍｉｎ）−０／１００（２０ｍｉｎ）−０／１００
検出波長：２５４ｎｍ As a result of measuring the delay time of the gradient from FIG. 11, it was 0.9 to 3.3 min, which was in a range having no practical problem. (Table 5)
This was smaller than a splitter using an FS silica tube as a resistor. This is a result of the dead volume being reduced because the monolith resistor can obtain a high pressure resistance even if it is short.

The reproducibility of the gradient was evaluated using the splitter 15 shown in FIG.
A pump (MP711) and a UV detector (MU701) were connected to the nanoflow splitter, and a resistor 22 side connection was used for evaluation. The flow rate on the column side during the evaluation was 0.16 μL / min. As shown in FIG. 12, it was found that there was high reproducibility (N = 3) even in a minute flow rate range of 0.16 L / min.
This proves that the monolith resistor produces a stable resistance.
Analysis conditions A. MeOH; B, 0.5% acetone (in MeOH)
A / B: 90 / 10- (5 min) -90 / 10- (15 min) -0/100 (20 min) -0/100
Detection wavelength: 254 nm

Since the resistor of the present invention can obtain a high pressure even if it is short, it can be directly connected to a three-way joint or the like, and can be easily introduced into different devices with a very small target flow rate by branching.
The resistor 3A having a sulpore of 1 μm in Example 1 was cut to a length of 10 mm and directly attached to the three-way joint 23. The same resistors 50 and 51 were attached to both ends, and the fluid was divided in half and introduced into different electrochemical detectors 24 and ultraviolet detectors 25. (Fig. 13)
Sample obtained by adding vitamin C to urine at 3 μL / min with 0.1 M potassium phosphate + 1 mM EDTA, aqueous solution (pH 3.0) using a column of Inertosyl (registered trademark) ODS-3, inner diameter 0.3 mm, length 150 mm Were analyzed simultaneously with ECD (200 mV) 24 and UV (254 nm) 25.
It was confirmed that 1.5 μL / min was uniformly introduced into each detector.
The monitoring result with UV is shown in FIG. 14, and the monitoring result with ECD is shown in FIG. Vitamin C component elution was observed at a retention time of 5.5 min.
As a result of the analysis, there was no change in the peak shape due to the internal capacity of the resistor, and each detector was detected at the same time, so even with a flow rate of a very small region of 3 μL / min, it was branched with good reproducibility. It was confirmed that the liquid was not affected.
As described above, it has been proved that the branching device using the resistance tube of the present invention is effective when branching to different detection of biological sample components that require analysis in a very small amount.

Since the resistor of the present invention can obtain a high pressure even if it is short, it can be directly connected to a three-way joint or the like, and a very small target flow rate can be easily obtained by branching.
For example, it is possible to connect a resistance tube directly to the commercially available three-way joint 23 in FIG. 16, branch, introduce a part into the detector 26, and collect a necessary part while monitoring it.
The resistor 1 </ b> A in Example 1 was cut to a length of 10 mm and directly attached to the three-way joint 23 as the resistor 42. On the detection side, the resistor 3A was cut to a length of 20 mm and directly attached to the three-way joint 23 as a resistor 43. The tip of the resistor 43 was introduced into the detector 26 via a flexible Teflon (registered trademark) tube 27. The three-way joint 23 was directly attached to the fraction collector 28 and installed so as to collect the outlets in a divided manner. Divided into 30 to 1 and partly collected with partial detection.
The resistor 1A has a sulpore of 2 μm, and the resistor 3A has a sulpore of 1 μm.

Inertsil (registered trademark) WP300Diol, an inner diameter of 0.5 mm, a length of 250 mm, and a 0.2 M sodium phosphate solution (pH 6.8) at 10 μL / min. Conalbumin (72 kDa), 2. 2. bovine serum albumin (66 kDa); Each fraction was collected with a fraction collector while monitoring the mixed protein of myoglobin (14 kDa). The sample was taken for 10 seconds at each peak top.
FIG. 17 shows an example of monitored results. Results with good reproducibility were obtained.
Each of the collected components was confirmed with 13% concentration acrylamide electrophoresis gel for the decomposition state of the components of each fraction.
FIG. 18 shows an electrophoresis photograph. One lane is a standard marker, two lanes are mixed components, and three, four, and five lanes are fraction components corresponding to 1, 2, and 3 in FIG.
As a result, even a trace amount was collected with good reproducibility, and it was confirmed that the resistance tube of the present invention had no adsorption. Furthermore, it was confirmed that there was no degradation of the collected protein, and there was no denaturation through a trace amount of easily degraded protein.

  In a mass spectrometer, reproducible ionization is important for high sensitivity analysis. In order to perform ionization with good reproducibility, introduction at a low flow rate is necessary. Therefore, branching to a minute flow rate to 1 μL / min or less is necessary. Furthermore, it is necessary to make the droplets as small and uniform as possible into the ionization chamber, and it is used by connecting a sprayer that is tapered to a diameter of several to several tens of microns. Tapering with good properties is difficult, reproducibility cannot be obtained, and yield is poor. Also, the sprayer itself is difficult and expensive to process. Since the monolith resistor of the present invention has a uniform spore of several to several tens of microns, uniform droplet formation is possible, and a directly branched liquid is introduced without connecting a special sprayer as in the prior art. be able to. However, mesopores must be monoliths of less than 2 nm because the presence of mesopores inhibits droplet formation. Furthermore, in order to facilitate ionization of the completed droplet, the outer diameter is desirably 0.2 mm or less.

In the same manner as in Example 1, methyltrimethoxysilane, formaldehyde as a coexisting substance, and 1 mol / 1 nitric acid aqueous solution as a catalyst were mixed at a molar ratio of 1: 1.8: 2.8 to obtain a uniform solution. .
Put this in a fused silica tube with an outer diameter of 0.15 mm, an inner diameter of 0.15 mm, an inner diameter of 0.05 mm, and a length of 5 m, and seal both ends with a polypropylene seal film, and gel at 40 ° C. for 15 hours. After the formation, the product was washed with 2 mL of methanol to obtain a resistor. This resistor was cut into 5 cm, and the resistor 12 was connected to the ionization chamber of the mass spectrometer 28.
To the resistor 13, the resistor 1A having a sulpore of 2 μm prepared in Example 1 was attached. (Fig. 19)
Using an Inertsil (registered trademark) ODS-3 3 μm HPLC column with an inner diameter of 0.3 mm and a length of 150 mm, a flow rate of 6 μL / min from a 20% acetonitrile aqueous solution containing 0.1% formic acid to 60% acetonitrile containing 1% formic acid Analysis of tryptic digests of β-casein was performed with a 40 minute gradient into aqueous solution. The UV side wavelength was 210 nm, and the mass spectrometer 28 detected with Nano ESI Plus.
The fluid having a flow rate of 6 μL / min was reproducibly branched to 5 μL / min for the UV detector 25 and 1 μL / min for the mass spectrometer 28 corresponding to the resistors 12 and 13.
Furthermore, it was ionized and analyzed with high sensitivity by the effect of the resistor 12 without a special sprayer. (FIG. 20) is the result of the UV detector, and FIG. 21 is the result of the mass spectrometer. With the monolithic resistor capable of reducing the capacity, detection was possible without delay even after branching.

  In the fields of biotechnology and pharmaceuticals, there is an increasing need for structural or functional analysis of proteins in various biological samples using two-dimensional or multidimensional LC / MS-MS. Separation techniques such as ion exchange, size exclusion, and reverse phase chromatography are combined according to the purpose and used as a two-dimensional or multidimensional LC. Each target component in the complex biological material is eluted from the final stage column in the two-dimensional or multi-dimensional LC system, and then branched to the downstream side through the split means. In doing so, one of the streams A is split into a measuring means (eg ESI-MS) and the other stream B is split into a fraction collector via a UV-VIS detector. Stream B is fractionated into a well plate or MALDI plate by a fraction collector. Each target component sorted into the well plate is further subjected to various post-processing for structural / functional analysis. Each target component fractionated on the MALDI plate is co-crystallized with the added matrix, and both plates are placed on the MALDI-TOF and MS for structural analysis. (Fig. 21)

The resistor 2A of Example 1 having a length of 20 mm was attached to 13, and the resistor 3A of Example 1 having a length of 14 mm was attached to 12. (Fig. 21)
A two-dimensional LC was assembled with Inertsyl (registered trademark) CX having an inner diameter of 0.3 mm and a length of 10 mm as the first column and Inertsyl (registered trademark) peptide C18 having an inner diameter of 0.3 MM and a length of 150 mm as the second column.
The salt concentration was changed in the first column, separated by pKa, and a reverse phase gradient was performed in the second column. The second column analysis conditions were a flow rate of 1.4 μL / min, and a 30 minute linear gradient analysis from 25% acetonitrile aqueous solution containing 0.1% formic acid to 40% acetonitrile aqueous solution containing 0.1% formic acid was performed. .
Due to the effect of the resistor of the present invention, it was reproducibly introduced at 0.4 μL / min on the mass spectrometry 28 side and 1 μL / min on the Mardi plate 29 side. As a result of adding matrix α-cyano-4-hydroxycinnamic acid (CHCA) 10 mg / 0.3% TFA from 30 and analyzing peptides containing Angiotensin I, the results of the ultraviolet detector 25 are shown in FIG.
FIG. 23 shows the MALDI-TOF-MS result of the eluate not passing through the resistor of the present invention, and FIG. 24 shows the result of MALDI detection of the portion of Angiotensin I eluting from 11 to 12 minutes.
The resistor of the present invention having a mesopore of less than 2 nm prepared without adding a surfactant or the like used for creating a mesopore is divided into an optimal flow rate with the same background and reproducibility as a solution blank, and is applied to the MADI-plate. We were able to prove that
Moreover, as a result of attaching 13 resistors directly to the tip portion spotted on 29, the same effect was obtained.
Since the resistor of the present invention can obtain a high pressure even in a small amount, a reproducible branch can be obtained even if another inlet such as addition of a matrix is provided between the resistors.

Application to the actual analysis using a splitter was performed.
The chromatograph shown in FIG. 25 has the same linear velocity with the same biological protein mixed sample and the same gradient program using the analysis column with the inner diameter of 200 μm from the bottom, the monolith type column shown in Table 3, the inner diameter of 100 μm, and 50 μm. It is the result of analyzing at the flow velocity which becomes. Both column lengths are 150 mm. The actual flow rate for the 200 μm inner diameter column is directly pumped from the pump at a flow rate of 2.0 μL / min without using a sputter, and for the 100 μm inner diameter column is 3 μL / min from the pump. The liquid is branched using a splitter and is set to be fed to the column at 0.50 μL / min. In the column with an inner diameter of 50 μm, 2 μL / min from the pump is branched using the splitter. And 0.15 μL / min was set to the column.
Analysis conditions Solution: A; H 2 O (0.1% TFA), B; CH 3 CN (0.1% TFA)
A / B = 80 / 20− (30 min) −50/50 (40 min) −50/50
Even when compared with direct pumping from the pump, the chromatogram using the splitter (upper two) showed the same elution pattern.
This indicates that the analysis was performed under the same linear velocity and the same gradient conditions in all analyses, and that the set flow rate could be split accurately by the splitter.

As shown in FIG. 26, on the column side after the branching tool, resistors having different resistance values obtained by adjusting the sulpore system and the inner diameter are installed in parallel as column-side resistors (internal resistors) 31-34. Connect the column to a resistor according to the purpose of use, and close the flow path for other resistors, or connect a sufficiently large resistor to the resistor and the waste liquid side. Splitting according to the ratio can be performed.
It is also possible to connect each resistor via a rotary valve 35 (one inlet (outlet) and plural) and select a single resistance tube by switching the valve. In this way, it is not necessary to close the flow path, and it is possible to easily select a resistance tube suitable for the purpose of use (FIG. 27). A rotary valve has a single inlet (outlet) and a plurality of outlets (inlet), and is a flow path switching device that can be selected by rotating a flow path connecting the inlet and the outlet.

For example, in Example 1, the mixing ratio of formaldehyde is changed to 0.3, 0.8, 1.3, 1.8, and 2.5, and sulpore 20 μm, sulpore 10 μm, sulpore 5 μm, sulpore 2 μm, sulpore 1 μm. A monolithic resistor is manufactured.
4 is a resistor having a sulpore of 5 μm, 31 is a resistor having a sulpore of 2 μm, 32 is a resistor having a sulpore of 5 μm, and 33 is a resistor having a sulpore of 1 μm. Therefore, when 31 lines are used, a flow rate divided by 6: 1 is obtained for 4 lines.
Similarly, the flow rate is divided into 1: 1 for 32 lines, 1: 4 for 33 lines, and 1:16 for 34 lines.
Also, as shown in FIG. 28, by connecting rotary valves 35 and 36 synchronized to both the upstream and downstream sides of the resistance tube connected to the analysis column, a single flow path for connecting the analysis column downstream of the resistance tube can be obtained. Therefore, it is not necessary to reconnect the column to match the resistance tube. This can reduce a troublesome operation particularly when connecting a column having a small diameter.

Further, as shown in FIG. 29, by using a recycle valve 37 (in the figure, two resistances are selected from three types of resistance pipes and are used as a waste liquid side and a column side), three types (or more) are used. By selecting any two types of resistance tubes from the resistance tubes 38-40, a more flexible split ratio can be created.
Similarly, when a resistor having a sulpore of 10 μm is used for 38, a resistor having a sulpore of 2 μm is used for 39, a resistor having a sulpore of 1 μm is used for 40, and the lines 38 and 39 are used, a flow rate divided by 25: 1 is obtained.
In the case of using 39 and 40 lines, a flow rate divided by 1: 4 is obtained.
As described above, by combining a monolith structure having different sulpores and various valves, the flow rate can be automatically switched.

The viscosity of liquid changes with temperature. Even in the splitter, the change in the viscosity of the liquid in the resistor body changes the solution feeding resistance, that is, the flow velocity to the column changes. When fused silica is used, since the liquid in the resistance tube is large, the change in viscosity due to a change in ambient temperature increases, and the split ratio is likely to shift. On the other hand, when the monolith-filled tube is used as a resistance tube, the amount of liquid in the resistance tube can be suppressed to a small amount, so that the influence can be suppressed to the minimum even if it is affected by the temperature.
The change of the flow rate by the temperature of this-article product Example 4 is shown.
The splitter apparatus was placed in an oven, the temperature was controlled at 10 ° C., 23 ° C., and 40 ° C., a 20% acetonitrile aqueous solution and water were fed, and the flow rate at the column side outlet was measured. (Table 6)
As a result, a fluctuation of 0.005 μL / min was confirmed for both the 20% acetonitrile aqueous solution and water. However, this fluctuation value is a fluctuation of about 1% of the operating flow rate, and is a fluctuation that can be sufficiently ignored for actual analysis.

A method for more accurately controlling the flow rate sent to the column after branching is shown below. (Fig. 30)
In the split mechanism as shown in the second embodiment, a flow rate sensor 41 capable of measuring the flow rate of the liquid delivered from the column is installed. When the flow rate measured by the sensor is not the proper flow rate of the column, it is possible to stably supply the optimal flow rate to the column by adjusting the amount of liquid fed from the pump by feedback from the sensor to the pump. .

Schematic explanatory diagram of conventional equipment Schematic explanatory diagram of conventional equipment Cross-sectional SEM photograph of resistor of one embodiment of the present invention Same as above Cross-sectional SEM photograph of resistor according to another embodiment of the present invention Same as above Cross-sectional SEM photograph of resistor according to another embodiment of the present invention Same as above Schematic illustration of splitter according to one embodiment of the present invention Performed chromatogram according to the above example Other embodiment of the present invention splitter schematic explanatory diagram FIG. 8 is a schematic explanatory diagram of the specific embodiment. FIG. 8 is a schematic explanatory diagram of the specific embodiment. The chromatogram according to the embodiment of FIGS. Chromatogram according to the embodiment of FIG. Other schematic illustration of the present invention Chromatogram according to the above example Chromatogram according to the above example Other schematic illustration of the present invention Chromatogram according to the above example Same as above Example electrophoresis photo Other schematic illustration of the present invention Same as above Example Chromatogram by UV detector Same as above Example Chromatogram by mass spectrometry Other schematic illustration of the present invention Same as above Example Chromatogram by UV detector Same as above Example Chromatogram by MALD-TOF-MS Example as above Chromatogram by MALDI detector The chromatogram according to the embodiment of FIGS. Other embodiments of the present invention Other embodiments of the present invention Other embodiments of the present invention Other embodiments of the present invention Other embodiments of the present invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Column 12 Waste liquid side resistance 13 Column side resistance 14 Connector 15 Splitter device 16 Base pipe 17 Waste liquid side resistor 19 Monolith side resistor 15 Connector

Claims (5)

Using a monolith structure having a continuous hole penetrating from 0.5 μm to 100 μm from the inlet to the outlet as a resistor, and branching the flow of liquid or gas to obtain a desired flow velocity corresponding to the monolith structure A fluid control method having a minute flow rate. The fluid control method for micro flow rate according to claim 1, wherein mesopores of the monolith structure are less than 2 nm. A device that obtains a desired flow rate by branching a liquid or gas flow , and penetrates 0.5 μm to 100 μm from the inlet to the outlet in the flow path after branching at the branching part or branching tool of the device . A fluid control apparatus comprising a resistor made of a monolith structure having continuous holes. The fluid control device according to claim 3, wherein mesopores of the monolith structure are less than 2 nm. 5. The fluid control apparatus according to claim 3, wherein the resistor is created in a flow path having an outer diameter of 0.2 mm or less connected to an analytical instrument such as a mass spectrometer.

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