JP3783055B2 - Microtrap screening method and apparatus - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention is a technology belonging to the field of analytical chemistry, and a sample is applied to a separation analyzer such as gas chromatography (GC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), thin layer chromatography (TLC), etc. As a pretreatment for supply, it relates to technology for extracting and concentrating target components from samples sampled from specimens, especially in the medical field where high-precision analysis results are required from extremely small samples such as samples from living organisms. The present invention also relates to a technology that can contribute to achieving these by simple means.
[0002]
[Prior art]
When qualitative / quantitative analysis is performed with a separation analyzer such as GC, HPLC, CE, or TLC, it is necessary to extract, purify, concentrate, etc. the sampled sample once as the pretreatment. There are many cases.
For these pretreatment methods, various methods corresponding to the target component have been used for a long time, but in recent years, the solid phase extraction (SPE) method, the headspace (HS) method, the purge trap (PT) method, A phase microextraction (SPME) method or the like is used.
[0003]
[Non-Patent Document 1]
“Latest separation, purification and detection method”, p94-104, NTS, 1997.
[Non-Patent Document 2]
Nara Ryoaki, Yashiki Mikio, Kojima Satoshi, Fukunaga Tokuto, Solid Phase Micro Extraction (SPME) Method in Forensic Toxicology, Forensic Poisoning, 16, 1-16, 1998.
[Non-Patent Document 3]
Japan Society for Analytical Chemistry Gas Chromatography Research Roundtable, “Capillary Gas Chromatography” p66-79, Asakura Shoten, 1997
[0004]
[Problems to be Solved by the Invention]
These conventional methods have various advantages when separating and extracting a dilute target component in a sample from a substance that interferes with the analysis, concentrating it, and then introducing it into an analyzer. However, the following disadvantages are present. There is.
That is, the disadvantages of the SPE method are that it is limited to liquid samples, is difficult to apply to the analysis of low-boiling compounds, and when introduced into an analyzer, the concentrated and collected target components are used. Since it is desorbed by using about 1 to 10 ml of solvent, it is possible that the concentrated components are diluted.
Further, the disadvantage of the HS method is that it is limited to the analysis of relatively low boiling point compounds, and in the case of classical operation, the concentration efficiency is low, so that it is inappropriate when the target component is an extremely dilute concentration. In addition, in order to compensate for this, when using a separate apparatus having a cooling or reconcentration function, the equipment becomes expensive and the operation becomes complicated.
[0005]
The disadvantages of the PT method can be applied to higher boiling point compounds compared to the HS method, but the equipment and operation are complicated, and some of the samples and specific components used last time are trap tubes or equipment piping. Etc., and carryover occurs, which often adversely affects the next analysis.
Furthermore, the disadvantage of the SPME method is that the carry-over problem at the time of repetitive analysis has not been solved while compensating for the disadvantages of the above methods, and the extraction efficiency is relatively low. It is easy to receive.
[0006]
In addition, these various pretreatment methods are used without much distinction between precision analysis and screening analysis, and screening analysis that requires rapidity, simplicity, and economy does not necessarily meet the requirements. .
SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to solve the above-mentioned problems including carryover while maintaining the advantages of the above-described conventional preprocessing operations.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, the present invention has the following configuration.
The microtrap screening method according to claim 1 is a microtrap screening method in which a sampled sample is extracted from the sample as a pretreatment for supplying the sampled sample to an analyzer, and screening is performed using a disposable microtrap tube. It is characterized by that.
The microtrap screening method according to claim 2 is characterized in that, in the method according to claim 1, a commercially available capillary column for gas chromatography is cut into a predetermined length as the disposable microtrap tube. .
The micro-trap screening apparatus according to claim 3, wherein the sample is enclosed in a container constituted by at least two phases, gas supply means capable of supplying gas into the one phase, and the gas supplied It is characterized by including a trap means having a microtrap tube to be disposable, capable of introducing a substance constituting a phase different from one phase.
The micro-trap screening apparatus according to claim 4, wherein a sample is enclosed in a container constituted by at least two phases, gas supply means capable of supplying gas into the one phase, and the gas supplied And a trap means having a disposable micro trap tube into which a substance constituting one phase can be introduced.
[0008]
According to a fifth aspect of the present invention, there is provided a micro trap screening apparatus including a trap means having a disposable micro trap tube and a sample introduction means capable of introducing a sample into the micro trap tube.
The micro trap screening apparatus according to claim 6 is the apparatus according to any one of claims 3 to 5, wherein the micro trap tube is formed by cutting a capillary column for gas chromatography into a predetermined length. It is characterized by being made.
The micro trap screening device according to claim 7 is the device according to any one of claims 3 to 6, wherein the trap means includes a micro trap tube and a needle portion connected to an end thereof. It is characterized by being configured.
[0009]
The micro-trap screening apparatus according to claim 8 is the apparatus according to any one of claims 3 to 6, wherein the trap means includes a micro-trap tube and a stainless tube externally provided on the micro-trap tube. It is characterized by that.
A microtrap screening apparatus according to a ninth aspect is the apparatus according to any one of the third to eighth aspects, further comprising a container heating unit capable of heating the container.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
The container used in the apparatus of the present invention is not particularly limited as long as it is a container that can enclose a sampled sample. Further, the container can be suitably used, for example, a vial with a septum, which is composed of a main body and a cap. However, the present invention is not limited to this. In particular, in the invention according to claim 5, the container itself is a syringe or the like. It can be configured with.
When the trap means or the gas supply means is connected to the container, it can be easily performed by penetrating the septum in a vial with a septum.
The gas supply means supplies an inert gas or the like to one phase inside the container composed of at least two phases by putting a sample, and supplies the target component contained in the gas phase part or the liquid phase part to the trap means. For example, a syringe provided with a needle can be exemplified. When the container is a vial with a septum, it is possible to easily connect to the container by inserting gas into the syringe and then penetrating the septum of the container with the needle. Further, when sampling and pretreatment are performed outdoors, a portable small cylinder or the like can be used as a gas source. As the gas source, various gas sources can be used as appropriate. The gas source is connected to the container directly or via a pipe and a needle-like part, and the gas is intermittently or continuously supplied into the container. Supply. As the gas to be supplied, an inert gas such as nitrogen or helium is generally used, but various gases may be used depending on the target component.
[0011]
Moreover, although the trap means has a micro trap tube, since this is made disposable, in the present invention, the carry-over problem which has been a problem in the conventional apparatus can be solved. In order to make the disposable more realistic, it is preferable to use the capillary column for gas chromatography cut into a predetermined length. The reason is that it is easy to ensure the uniformity of the performance of individual micro trap tubes and the economic efficiency. The capillary column for gas chromatography has a standard length of 15 to 60 m, and the uniformity inside thereof is quality controlled, and is indicated by numerical values such as coating efficiency. Therefore, even when a part of a capillary column is cut and used individually, variations in the amount and nature of the partition phase or adsorbed phase for extracting the sample substance are the results required for the efficiency of the sample extraction and the screening analysis. It is negligible from the reproduction accuracy. Although such a commercially available capillary column itself is expensive, the length of the micro trap tube used in the apparatus of the present invention is appropriately set according to the sample, the target substance, and the like, and is approximately several centimeters level (for example, 0.5 cm). ˜100 cm, preferably 5 cm to 10 cm), a large number of microtrap tubes can be obtained from one capillary column, which is sufficiently economical. This makes it possible to obtain a trap tube having the required level of uniformity at a very low cost.
[0012]
As a material of the micro trap tube, a fused silica tube, a borosilicate glass, a stainless tube, or the like can be used, which is also a material of a general GC capillary column that is commercially available. In the case of a micro trap tube formed from a capillary column made of fused silica, if there is a possibility that the outer surface of the tube may come into contact with the sample, a film (polyimide resin film, It is preferable to remove the aluminum thin film, etc., and in the case of a stainless steel pipe, in order to avoid the target substance being adsorbed by the constituent metal and the catalytic action of the metal, the inner and outer surfaces of the pipe are not removed. It is preferable to perform an activation treatment.
Prior to use, it is preferable to reduce the background and make each trap tube uniform by heat-treating a plurality of them simultaneously under an inert air stream. The internal diameter of the column suitable for use in the present invention is, but not limited to, 0.1 to 1.2 mm, preferably 0.2 to 1.0 mm.
In addition, an internal standard, salts, an anticoagulant, a reagent for promoting the dissociation of the conjugate, and the like may be added to the container together with the sample as necessary.
The trapped component is desorbed by heating, a trace amount of solvent, and the like, introduced into an inlet of an analyzer such as GC, HPLC, CE, TLC, etc., and used for analysis.
[0013]
When the trap means includes a micro trap tube and a needle portion such as an injection needle connected to the tip, the septum of the container can be penetrated and connected by the injection needle, and the tube and the needle portion are connected. The portion is preferably a Teflon chip or the like. The needle can also be used to penetrate the inlet septum of GC or the like, and is also used when feeding the desorbed sample to the GC or the like.
In addition, when the trap means is configured to include a micro trap tube and a stainless steel tube sheathed on the micro trap tube, the stainless trap tube at the downstream end of the introduction flow of the micro trap tube has a small inner diameter to prevent the micro trap tube from coming off. It is preferable to make it a shape. In this case, the septum can be penetrated by the stainless tube without connecting the needle part. Similarly to the above, it can be used for penetration of an inlet septum such as GC.
Furthermore, it is possible to penetrate the septum with the microtrap tube itself.
Note that the fluid that has passed through the micro trap tube is discharged from the end portion on the side opposite to the sample introduction side, but when the fluid is a gas, the end portion may be opened as it is to be discharged into the air. Piping can be connected and collected regardless of the liquid.
[0014]
When the apparatus of the present invention further includes a container heating means, it is possible to more easily volatilize a substance that does not volatilize and increase the extraction rate. It should be noted that stirring with a stirrer bar in the container also enables efficient extraction. As specific examples of the container heating means, various heating devices generally used in the industry can be used.
The micro trap tube in which the target component is trapped is placed in a sealed container, and is kept under temperature control as necessary, and is stored, transported, and the like.
[0015]
【Example】
Hereinafter, the present invention will be specifically described with reference to the case where a liquid sample is sampled.
Please refer to FIG. A sample 5 (liquid) is sealed in a vial 3 having a septum 1 and a sealing cap 2, leaving a gas phase portion 4 of the upper space (head space). An injection needle 7 penetrating the septum 1 is connected to the distal end of the gas pipe 6 constituting the gas supply means, and the distal end of the injection needle 7 is immersed in the sample 5. Further, the micro trap tube 8 constituting the trap means itself penetrates the septum 1 and opens its lower end into the gas phase portion 4. The penetration of the septum 1 may be performed by an injection needle (not shown) connected to the lower end of the micro trap tube, or may be performed through an externally mounted stainless steel tube (not shown). The upper end of the micro trap tube may remain open or may be configured to collect the fluid that has passed by connecting a pipe (not shown) or the like, depending on the properties of the sample.
In this example, since the tip of the injection needle 7 has reached the inside of the liquid phase of the liquid sample, for example, when a small amount of inert gas is continuously introduced, the target component dissolved in the liquid is purged. The gas phase is transferred to the gas phase section 4 and further introduced into the micro trap tube, and gas / liquid distribution (or gas / solid adsorption) is held and concentrated in the stationary phase made of liquid (or solid) on the inner surface of the micro trap tube 8. The
After completion of extraction, the microtrap tube can be removed from the container, placed in a sealed container according to the nature of the sample, and transported to a facility with an analyzer under appropriate temperature control.
[0016]
Please refer to FIG. The same members as those in FIG. In this example, the lower end of the injection needle 7 constituting the gas supply means opens to the gas phase portion 4. Therefore, by continuously introducing the purge gas, the target component in the gas phase portion 4 and the target component transferred from the liquid to the gas phase portion are continuously introduced into the micro trap tube based on the principle of partial pressure equilibrium, It is similarly concentrated in the trap tube.
About the conveyance after extraction, it is the same as that of the above.
[0017]
Please refer to FIG. The same members as those in FIG. The injection needle 7 opens in the gas phase part 4 as in the example of FIG. 2, and the lower end of the microtrap tube 8 is immersed in the sample 5.
In this example, the liquid sample is directly introduced into the microtrap tube by introducing gas, and the target component is concentrated in the microtrap tube by liquid / liquid distribution (or liquid / solid adsorption). In the figure, the shape of the inner surface of the container bottom is an inverted conical shape so that the liquid sample can be efficiently introduced into the micro trap tube.
As a convenient method, it is also possible to sample a fluid sample regardless of gas or liquid into a syringe and press-fit it into a microtrap tube at a slow speed.
[0018]
In the introduction to the analyzer, the introduction into the GC is performed by heating the micro trap tube and desorbing the extracted substance. In this case, a method is generally used in which an inert gas having an appropriate flow rate is introduced from the anti-GC side of the micro trap tube and heated, and the extracted substance is thermally desorbed and introduced into the GC. Alternatively, by sealing the anti-GC side of the trap tube and heating the trap tube by an appropriate method, the substance extracted in the trap tube is desorbed, and most of the GC is removed by the pressure due to the heated volume expansion of the vaporization section. It is also possible for convenience to introduce.
In addition, in the case of a substance having a high boiling point or a non-volatile substance, the analysis is performed by HPLC. In this case, a syringe needle suitable for an HPLC sampling valve is provided on the HPLC side of the microtrap tube, and a microsyringe is provided on the anti-HPLC side. A base adapted to the syringe needle attachment part is attached, and the microtrap tube is left for a certain period of time with the desorption solvent filled, and then the syringe plunger is pushed in to introduce the desorbed sample together with the solvent into the sampling valve. In this case, since a very small amount (for example, 10 μL or less) of the desorption solvent is sufficient, it is not diluted as much as the conventional method and does not hinder the microanalysis. In addition, using a loop sampler with a 6-way valve, a pressure-resistant resin is used for the loop, and a microtrap tube is inserted into the loop for solvent filling and desorption, and introduction into the HPLC by switching the valve. It can also be done.
[0019]
【The invention's effect】
According to the present invention, the following effects can be obtained.
Since the present invention is a dynamic extraction and uses a micro trap tube, the extraction efficiency can be greatly increased, and a recovery rate of 100% is also possible. As a result, analysis with high accuracy is possible, and therefore, it can also be used for analysis of a specimen having a small sample amount such as a biological sample (necessary sample amount: less than 1 ml).
In the SPME method, as in the present invention, the trap member can be directly immersed in the liquid and can be extracted from the head space, but in any case, it is a static method. On the other hand, since the present invention is a dynamic extraction, it can be added in a large amount, and the SPME method directly exposes the extraction phase into the sample or the sample headspace, so that the volume ratio (phase ratio) between the sample and the extraction phase However, even when the partition coefficient of the extraction phase with respect to the target substance is large, the amount of the substance that is actually extracted is small, and the extraction efficiency is at most about 10 to 20%. Because the micro trap tube is used, the space volume in the trap tube is extremely small, and the phase ratio (sample space volume / extraction phase volume) in the distribution equilibrium is small. The amount of sample can be extracted, and when desorbing and introducing into the analyzer, the solvent is not desorbed or the amount is extremely small even if used, so that the desorbed sample does not diffuse, This is because it can be introduced into an analyzer such as GC. As a result, the extraction absolute amount increases, the spread of the sample band is reduced, and the S / N ratio is improved, so that relatively high sensitivity can be obtained (detection sensitivity: ppt level).
[0020]
In addition, in the SPME method and similar extraction methods, a method of increasing the film thickness of the extraction phase has been attempted in order to improve the recovery rate, but there is a limit to the increase ratio of the extraction phase volume to the sample volume. In addition, not only a large effect cannot be expected, but also it takes time to diffuse the sample material in the extraction phase, so that it is indispensable to increase the distribution equilibrium and extraction time. On the other hand, in the present invention, since the sample volume in the extraction part is greatly reduced, the film thickness of the extraction phase can be made smaller, thereby achieving distribution equilibrium and shortening of the extraction time.
[0021]
Furthermore, carry-over can be completely avoided by disposing the micro trap tube disposable.
In addition, the introduction into the analyzer enables selective desorption according to the desorption temperature by adjusting the heating temperature of the micro trap tube, and screening with a limited target component can be performed.
In the present invention, unlike the PT method, a large-scale apparatus is not required, and the structure and operation procedure are simple, so that even beginners can operate. Since the sample can be collected and concentrated at a location away from the device installation location, it is not necessary to transport the sample, and the sample can be collected and concentrated at the location where the sample is located. Therefore, not only can the labor of transferring the sample be saved, but also alteration during sample transfer can be prevented.
Furthermore, since it is not necessary to modify the analyzer such as GC, it is economical and convenient.
[0022]
Furthermore, when a GC capillary column is cut to a predetermined length and used, the column material and size, and the variety of stationary phases, are very selective according to the sample, and the tube length is also the test sample. Therefore, the versatility of the device is high. In addition, the quality is guaranteed, the uniformity of the micro trap tube can be ensured, and further economic efficiency can be obtained.
Moreover, since concentration collection by a liquid phase is also possible, in this case, it can be applied also to a high boiling point thing, and versatility is acquired.
In the case of having a heating device, it is possible to collect substances that are difficult to volatilize by heating the sample by the principle of steam distillation, and it can be applied from low boiling point compounds to high boiling point compounds. In addition, since the high molecular compound which comprises the biological body contained in a biological component cannot transfer in a trap pipe | tube via a gaseous phase also by heating or steam distillation, it is not collected. Further, even if other high molecular substances that are not suitable for GC analysis or HPLC analysis are collected, they remain in the trap tube at the time of desorption, so that the analysis is not hindered.
[0023]
According to the present invention, accuracy and confirmation according to the present invention can be improved by adding a surrogate compound in the sample or adding an internal standard to the desorbed solvent in accordance with a general analysis method. Concerns about tube reproducibility can also be wiped out by checking each time or periodically.
As described above, the present invention solves the problem of carryover, and further reinforces those weak points while maintaining the advantages of the SPE method, HS method, PT method, SPME method and the like that have been used conventionally. In addition, the target component can be easily and economically extracted from the sample specimen with high efficiency, and can be introduced into the separation analyzer by a simple method.
That is, in the apparatus of the present invention, the target specimen sample may be a gas, liquid, or solid, and from an extremely low boiling point compound to a high boiling point compound, and also a high molecular compound may be pretreated for analysis. Is possible.
In addition, extraction, concentration, and introduction into an analytical device can be performed from a very small amount of sample such as a biological sample without introducing coexisting substances in a sample sample that interferes with analysis into the analytical device. .
Therefore, the apparatus of the present invention is very useful in the analysis of biological samples, water quality tests, food component analysis, and the like at medical sites.
[Brief description of the drawings]
FIG. 1 shows an embodiment of the apparatus of the present invention, in which a gas is supplied into a liquid sample.
FIG. 2 shows an embodiment of the apparatus of the present invention and shows a case where gas is supplied to the gas phase portion.
FIG. 3 shows an embodiment of the apparatus of the present invention, and shows a case where a liquid is introduced into a trap tube.
[Explanation of symbols]
1 Septum, 3 vial, 4 gas phase, 5 liquid sample, 6 gas piping, 7 injection needle, 8 micro trap tube

Claims (6)

  A container that is composed of at least two phases by enclosing a sample, a gas supply means capable of supplying a gas into the one phase, and a substance constituting a phase different from the one phase supplied with the gas And a trap means having a microtrap tube which is made disposable by cutting a commercially available capillary column for gas chromatography.   By enclosing a sample, it is possible to introduce a container constituted by at least two phases inside, a gas supply means capable of supplying gas into the one phase, and a substance constituting the one phase supplied with gas. And a trap means having a disposable micro trap tube produced by cutting a commercially available capillary column for gas chromatography. The cut, microtrap screening device according to claim 1 or 2 wherein and performing at optimal length to test sample. The micro trap screening apparatus according to any one of claims 1 to 3, wherein the trap means includes the micro trap tube and a needle portion connected to an end portion of the micro trap tube. The micro trap screening apparatus according to any one of claims 1 to 3, wherein the trap means includes the micro trap tube and a stainless steel tube sheathed on the micro trap tube. The microtrap screening apparatus according to claim 1 , further comprising a container heating unit capable of heating the container.

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