JP3763012B2 - Micro sample injector - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a micro sample injector widely used in liquid chromatography. The present invention also relates to a micro sample injector that can be used in capillary electrochromatography, capillary electrophoresis, separation analysis and measurement fields that can introduce a micro sample.
[0002]
[Prior art]
Currently, the minimum sample injection volume for sample injectors used in liquid chromatography is 60 nanoliters. In liquid chromatography, it is generally accepted that about 1% or less of the stationary phase volume of the filler is appropriate and appropriate for the sample loading. Since it is generally difficult to estimate the stationary liquid phase of the separation column, it can be considered that an injection volume of about 0.1% of the separation column length is possible. Assuming that the sample injection volume is 0.1% of the column length in a column having a diameter of 1 mm, the column having a length of 10 centimeters is equivalent to 0.1 mm, and the space volume in the column is 0.078 microliters. As such, the possible sample injection volume is 78 nanoliters. Further, when a general packing material (packing density 0.40 g / ml, pore volume 0.87 ml / g) is filled in a space corresponding to 0.78 microliters, the pore volume in the column is 0. Since it is estimated to be 27 microliters, 0.1% of 27 nanoliters can be regarded as a possible sample injection volume.
[0003]
In a capillary column having an inner diameter of the separation column of 0.5 millimeters or less, the injection amount of the sample solution is 20 nanoliters, but there is no injector that can inject such a very small amount of sample.
[0004]
If a large amount of sample solution is injected, the proper loading of the sample on the column will be exceeded, so that the peak obtained on the chromatogram will be broadened.
[0005]
In capillary electrophoresis, a hollow tube having an inner diameter of 50 micrometers to an inner diameter of 100 micrometers is used as a separation column. In general, when the column length is 50 centimeters, the sample injection width is suitably 1 to 2 millimeters. The volume of 2 millimeters in length with an internal diameter of 50 micrometers is 4 nanoliters, and similarly for a column using a 100 micrometer internal diameter hollow tube, it is 16 nanoliters. Since this is the upper limit possible, it is actually desirable to have a smaller volume of sample injection.
[0006]
Although there have been cases where a sample injector has been used so far, this internal volume is as large as 0.3 microliters, so it is not suitable for a column having the above inner diameter. The injection volume used for capillary electrophoresis is 4 nanoliters to 10 nanoliters, but there is currently no such a small amount of sample injector.
[0007]
[Problems to be solved by the invention]
In the liquid chromatography using a capillary column, injection by the split injection method is performed. In this case, a part of the sample solution is injected into the column, and the other part is discharged and not used. The part is not being used effectively.
[0008]
In capillary electrophoresis, a valve-type micro sample injector has not been generally used. Instead, one end of the column is attached to the sample solution, and a sample is electrically introduced by applying a voltage to the sample solution for a certain period of time. An injection method by suctioning with a difference in height at both ends of the column, installed at both ends of the column An injection method is used in which one of the electrodes attached to the solvent tank is pressurized or sucked to guide the sample solution into the column from the solvent tank holding the sample solution. However, since these three injection methods cannot determine the absolute injection amount of the sample solution, there has been no choice but to use a method that always estimates the injection amount from an empirical rule.
[0009]
In addition, one of the problems with valve-type sample injectors is that the sample solution is completely filled in the internal rotor. Not happening. In the valve-type injector, it is an object to determine that the sample solution is completely filled without bubbles in the sample solution holding chamber inside the injector. This is particularly important in a small sample injector.
[0010]
Therefore, the present invention assumes that a very small amount of sample solution holding chamber is provided in the rotor inside the valve-type sample injector, and that the sample solution can be injected with a certain amount of sample reliably, and the sample injection The purpose of the present invention is to provide a micro sample injector capable of visually, visually and electrically confirming that a sample has completely entered the container.
[0011]
[Means for Solving the Problems]
Therefore, the micro sample injector of the present invention has a sample solution holding chamber having a very small inner diameter that cannot be machined by inserting a very small hole forming body F into the hole 12 formed by machining. 4 is provided with the rotor 1 provided.
[0012]
In the present invention, a hollow tube 13 having an extremely small hole 13a is used as the formed body F, and the hollow tube 13 is inserted into the hole 12 and fixed, or the formed body F is formed as an extreme. A fine metal wire 14 is used, and the metal wire 14 is inserted into the hole 12, and after filling the hole 12 with a resin and solidified, the metal wire 14 is chemically dissolved or mechanically pulled out. It is assumed. When the metal wire 14 is mechanically pulled out, the metal wire 14 and the solidified resin can be easily peeled off through an electric current.
[0013]
Furthermore, in the present invention, the rotor 1 can be made of a transparent resin so that it can be visually observed that the sample solution is introduced into the sample solution holding chamber 4 without air bubbles.
[0014]
In the present invention, an optical fiber for enabling imaging of the state of the sample solution introduction in the rotor 1 can be provided.
[0015]
Furthermore, in the present invention, means for measuring the electrical resistance in the sample solution holding chamber 4 for confirming the state of introduction of the sample solution in the rotor 1 can be provided.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows an arrangement when the micro sample injector of the present invention is used in a liquid chromatograph system, in which a is a liquid feed pump, b is a micro sample injector, c is a separation column, and d is a detector. is there. FIG. 2 shows an arrangement when the micro sample injector of the present invention is used in a capillary electrophoresis system, where b is a micro sample injector, c is a separation column, e is a high voltage power source, and f is a solvent bath. is there. FIG. 3 shows an arrangement when the micro sample injector of the present invention is used in an electrochromatography system, where a is a liquid feed pump, b is a micro sample injector, c is a separation column, and e is a high voltage power source. , F is a solvent tank.
[0017]
In the liquid chromatograph system, the capillary electrophoresis system and the electrochromatography system, as shown in FIGS. 4 to 6, the rotor 1 provided in the micro sample injector b of the present invention has the holding portions 2, 3 on both sides. Is held by. A mandrel S is attached to the rotor 1, and a sample solution holding chamber 4 and flow paths 5 and 6 are provided. The holding unit 2 is provided with a liquid feeding channel 7 and a sample solution introduction channel 8, and the holding unit 3 is provided with a sample solution introduction channel 10 to the tip 9 a of the separation column 9 and discharge of the sample solution. A working channel 11 is provided.
[0018]
In the micro sample injector of the present invention, the sample solution is introduced from the introduction channel 8 into the sample solution holding chamber 4, and then the rotor 1 is rotated by a fixed angle so that the sample solution holding chamber 4 is introduced into the feeding channel 7 and the introduction channel. When set so as to match the flow path 10, the sample solution is injected into the separation column 9.
[0019]
In such a micro sample injector, the rotor 1 is a sample having an extremely small inner diameter that is impossible by machining by inserting a very small hole forming body F into a hole 12 formed by machining. A solution holding chamber 4 is provided. Specifically, the sample solution holding chamber 4 is provided as described below.
[0020]
That is, as shown in FIG. 5, a hole 12 is drilled in the rotor 1 in advance. The hole 12 has a minimum diameter of 0.2 mm in the current technology, but a hole of 0.6 mm is formed as an example. Next, a hollow tube 13 having a very small diameter hole 13 a of 0.2 mm or less is inserted into the hole 12. As an example, the hollow tube 13 has an inner diameter of 0.06 millimeters and an outer diameter of 0.6 millimeters. The inserted hollow tube 13 is sufficiently fixed between the wall surface and the rotor 1 with a novolac resin adhesive. Next, both sides of the rotor 1 are finished. In this way, the sample solution holding chamber 4 having a volume of 12 nanoliters is provided. For the rotor 1, polyimide resin, polycarbonate resin, Teflon resin, or the like can be used. As the hollow tube 13, a polyether ether ketone, a Teflon tube or the like can be used.
[0021]
As shown in FIG. 6, a hole 12 having a diameter of 0.6 mm is drilled in the rotor 1, and a metal wire is used using a fixture (not shown) installed outside the rotor 1 in the center of the hole 12. 14 is installed, and then resin powder or a resin liquid is injected into the hole 12 and heated and molded. Thereafter, the metal wire 14 is pulled out, and finish polishing is performed again to provide a sample solution holding chamber 4 having a very small inner diameter. As an example, a metal wire 14 having an outer diameter of 0.05 mm is used. When the metal wire 14 is pulled out, a voltage is applied to the metal wire 14, and the resin and the metal wire 14 are peeled off by the generated Joule heat, so that the metal wire 14 can be easily pulled out. Alternatively, the metal wire 14 may be pulled out by dissolving the metal wire 14 using a hydrochloric acid solution, and similarly providing the rotor 1 with a sample solution holding chamber 4 having a very small inner diameter.
[0022]
Furthermore, the micro sample injector of the present invention has a hole for holding an optical fiber (not shown) in the vicinity of the sample solution holding chamber 4 in order to make sure that there are no bubbles in the sample solution held in the rotor 1. 1, an optical fiber (not shown) is inserted into the optical fiber holding hole, and the state of the sample solution holding chamber 4 can be imaged. In this case, the rotor 1 is made of a transparent resin. As an example, polycarbonate resin was used. An image of the sample solution holding chamber 4 obtained by the optical fiber can be captured by a CCD camera and displayed as an image on a CRT.
[0023]
Further, as a means for checking the state of the sample solution holding chamber 4, the electric resistance in the sample solution holding chamber 4 changes when bubbles are present in the sample solution holding chamber 4. Can be grasped. In the micro sample injector according to the present invention, a thin metal wire (not shown) inserted from the outside is provided in the inside of two flow paths, that is, the liquid supply flow path 7 and the introduction flow path 10, and this is used as an electrode. The holding portions 2 and 3 of the rotor 1 are used as metal plates, and these are used as electrodes, and an electric field is applied between these electrodes to measure electric resistance. If air bubbles are present in the sample solution holding chamber 4, the electrical resistance increases, so that the state in the sample solution holding chamber 4 can be determined.
[0024]
FIG. 7 shows a chromatogram when liquid chromatography is carried out by a chromatographic system using the micro sample injector of the present invention. The thickness of the rotor was 4.5 millimeters, and the inner diameter of the hollow tube was 60 micrometers. The analysis wavelength of liquid chromatography was 254 nanometers, the wavelength resolution was 5.0 nanometers, the analysis time range was 0 to 10 minutes, and the data interval was 0.50 seconds. From FIG. 7, the sample injection amount by the micro sample injector of the present invention is estimated to be 12.7 nanoliters, but by changing the thickness of the rotor or adjusting the inner diameter of the hollow tube inserted into the rotor. The sample injection amount can be changed.
[0025]
As a reference example, FIG. 8 shows a chromatogram when liquid chromatography is performed by a chromatographic system using a sample injector having a sample solution holding chamber with a minimum commercially available sample injection amount (60 nanoliters). It was. The analysis wavelength of this liquid chromatography was 254 nanometers, the wavelength resolution was 5.0 nanometers, the analysis time range was 0 to 16 minutes, and the data interval was 0.38 seconds.
[0026]
Although not shown, capillary electrophoresis or electrochromatography could be performed using the micro sample injector of the present invention.
[0027]
【The invention's effect】
Since the present invention is configured as described above, it was possible to provide a micro sample injector optimal for a chromatographic system using a capillary column as a separation column. And since the trace amount sample injector of this invention can introduce | transduce all the trace amount sample solutions into a separation column, it became possible to utilize a valuable sample solution effectively. Furthermore, the micro sample injector of the present invention is superior to the conventional one in terms of reproducibility, and the reliability of a chromatographic system using a capillary column is improved.
[0028]
In addition, valve-type micro sample injectors have not been generally used in capillary electrophoresis, but if the micro sample injector of the present invention is used for capillary electrophoresis, the absolute amount of sample solution to be injected can be determined. According to the present invention, a valve-type micro sample injector can be provided for the first time in capillary electrophoresis.
[0029]
One of the problems of the valve-type micro sample injector is that the sample solution is completely filled in the internal rotor. However, the micro sample injector is easily affected by mixing of bubbles and the like. The micro sample injector of the present invention can confirm whether the sample solution holding chamber in the rotor is sufficiently filled with the sample solution by visual observation or imaging with an optical fiber, and can also be confirmed from the electric resistance value. As a result, the stability of the operation can be greatly improved.
[Brief description of the drawings]
FIG. 1 is a block diagram showing an arrangement when a micro sample injector of the present invention is used in a liquid chromatograph system.
FIG. 2 is a block diagram showing an arrangement when the micro sample injector of the present invention is used in a capillary electrophoresis system.
FIG. 3 is a block diagram showing an arrangement when the micro sample injector of the present invention is used in an electrochromatography system.
FIG. 4 is a cross-sectional view showing a combined state of a rotor of a micro sample injector of the present invention, holding portions on both side surfaces thereof, and an internal flow path.
FIG. 5 is an explanatory view showing an example of how to provide a sample solution holding chamber in the rotor of the micro sample injector of the present invention.
FIG. 6 is an explanatory view showing another example of how to provide a sample solution holding chamber in the rotor of the micro sample injector of the present invention.
FIG. 7 is a chromatogram obtained by a chromatographic system using the micro sample injector of the present invention.
FIG. 8 is a chromatogram obtained by a chromatographic system using a conventional micro sample injector.
[Explanation of symbols]
1 Rotor 4 Sample solution holding chamber 12 Hole 13 Hollow tube 13a Hole 14 Metal wire F formed body

Claims (8)

A rotor provided with a sample solution holding chamber (4) having a very small inner diameter that cannot be achieved by machining by inserting a very small hole forming body (F) into the hole (12) formed by machining. A micro sample injector characterized by comprising (1). A hollow tube (13) having an extremely small hole (13a) is used as the formed body (F), and the hollow tube (13) is inserted into the hole (12) and fixed. The micro sample injector according to claim 1. After using the very fine metal wire (14) as the forming body (F) and inserting the metal wire (14) into the hole (12) and filling the hole (12) with a resin and solidifying it. The micro sample injector according to claim 1, wherein the metal wire (14) is chemically dissolved. After using the very fine metal wire (14) as the forming body (F) and inserting the metal wire (14) into the hole (12) and filling the hole (12) with a resin and solidifying it. The micro sample injector according to claim 1, wherein the metal wire (14) is mechanically pulled out. The micro sample injector according to claim 4, wherein, when the metal wire (14) is mechanically pulled out, the metal wire (14) and the solidified resin are easily separated through an electric current. 2. The trace amount according to claim 1, wherein the rotor (1) is made of a transparent resin, and it is possible to visually check that the sample solution is introduced into the sample solution holding chamber (4) without bubbles. Sample injector. The micro sample injector according to claim 1, further comprising an optical fiber for enabling imaging of a state of introduction of the sample solution in the rotor (1). The micro sample injector according to claim 1, further comprising means for measuring an electric resistance in the sample solution holding chamber (4) for confirming a state of introduction of the sample solution in the rotor (1).

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