KR20090030761A - Dual stacking method combining large volume single drop microextraction and sweeping and the capillary for large volume single drop microextraction - Google Patents

Abstract

A method for performing single drop microextraction is provided to form microdroplet steadily in capillary electrophoresis and concentrating the sample. A method for performing single drop microextraction by forming microdroplet at the end of the capillary tube for preconcentration of the sample in capillary electrophoresis, comprises a step for inserting hydrophobic tube at the end of the capillary tube to form single drop stably. The tube is selected from the group consisting of polytetrafluoroethylene, perfluoroalkoxy polymer resin, fluorinated ethylenepropylene and polyetheretherketones.

Description

Dual stacking method combining large volume single drop microextraction and sweeping and the capillary for large volume single in capillary electrophoresis drop microextraction}

The present invention relates to a capillary tube for the double concentration method and a large volume single drop micro-extraction of the sample combined with a large volume single drop microextraction method and sweeping method in capillary electrophoresis. More specifically, in the capillary electrophoresis, microdrops are formed at the end of the capillary tube to form liquid droplets in the microscopic extraction of liquid phase. It is a technique for preconcentrating a sample or an extremely low concentration of the sample to significantly increase the sensitivity in the analysis thereof.

Capillary electrophoresis (CE) is a separation method that can be usefully used in various fields, but it is difficult to analyze trace amounts due to its low sensitivity in absorbance detection. Capillary electrophoresis is an excellent method of separation with high efficiency and high degree of separation, but it has a disadvantage of low sensitivity due to the use of a small amount of sample in CE. Especially in the analysis of proteins and metabolites, methods for improving sensitivity for qualitative analysis of low concentration samples are required. The advantage of concentrating on the column is that it is possible without any mechanical modification of the column, and the concentration procedure is quite simple, with little manipulation of the buffer and sample system. However, this method has the disadvantage that it is applied only if it meets specific requirements for buffer and sample solutions. As an alternative to this method, the preconcentration method of the sample connected with CE may be used. As a result, liquid-liquid extraction (LLE) was applied to CE (Pedersen-Bjergaard, S. et. Al., J. Chromatogr., A 2000 , 902 , 91-105). Various liquid phase microextraction methods have been applied to CE that achieve higher concentration effect than LLE but consume less solvent. However, the experimental operation is very complicated, shows memory effect, and insufficient sensitivity improvement. have. Therefore, the problem of increasing the sensitivity by concentrating the sample has become a very important problem.

Single drop microextraction (SDME) can be used as a method of preconcentration of samples in capillary electrophoresis. By simply programming the sequences in which the samples are processed, a single drop of an aqueous acceptor phase coated with a thin layer of organic phase can form at the end of the capillary. The analyte can be preconcentrated by the pH difference between the aqueous acceptor phase and the aqueous donor phase. However, the method of concentrating a sample by making microdrops coated with an organic solvent in capillary electrophoresis is expected to increase detection sensitivity, but there is a problem that microdroplets leak through the organic solvent. In addition, when the extraction time is long or vigorous stirring is applied, there is a problem that the stability of the microdrops falls.

On the other hand, if the microdroplets can be formed more stable and large, the concentration efficiency may be higher. If larger droplets can be produced in experiments that require a large amount of sample injection, the application of CE will be extended and it will be easier to link with other concentration methods.

An object of the present invention is to provide a means for improving the sensitivity by concentrating a sample in capillary electrophoresis. In making microdroplets in the capillary tube and concentrating the sample, the problem of not easily forming the microdroplets or deteriorating the stability of the formed microdroplets is a problem to be solved first. In addition, if a single volume micromeasurement (large volume SDME) having a large volume is possible, the scope of application of the single droplet microextraction method can be broadened, and on the basis of this, it is intended to provide a double concentration method in combination with the sweeping method.

The present invention for this purpose is to form a single drop at the end of the capillary for preconcentration of the sample in capillary electrophoresis in the method of performing a single drop microextraction, the end of the capillary tube can be formed stably It characterized in that the hydrophobic tube is inserted into the tube is polytetrafluoroethylene (PTFE), perfluoroalkoxy polymer resin (PFA), fluorinated ethylene-propylene (fluorinated ethylene-propylene; It is preferably selected from the group consisting of Teflon tubes such as FEP) and polyetheretherketones (PEEK) tubes.

In addition, the present invention is a method for preconcentration of the sample in capillary electrophoresis, again sweeping the sample concentrated by a single drop micro-extraction method (single drop micro-extraction-sweeping method; SDME-sweeping method) A method of preconcentration of a sample, characterized in that the re-concentration process using, the sweeping method, micelle electrokinetic chromatography (MEKC) having analyte sweeping effect after a single drop microextraction It is desirable to collect and accumulate in the pseudostationary phase (micelles) by the method. This includes forming a single drop at the capillary end of the capillary electrophoresis apparatus; Concentrating the sample in the single drop; Injecting the sample concentrated in a single drop into a capillary; Sweeping the sample injected into the capillary; preconcentration of the sample in capillary electrophoresis comprising a single drop of capillary end selected from the group of tubes and inserted into the end of the capillary, It is preferred to further comprise the step of stably forming a single drop.

The present invention provides a simple means capable of stably forming microdroplets in liquid phase microextraction by forming microdroplets at the end of capillary electrophoresis in capillary electrophoresis and forming a large volume. In addition, large volume single droplet micromembrane (Large volume SDME) can be used to extend the scope of single-drop microscopic extraction, such as sweeping or field-amplified sample stacking (FASS) It is easier to carry out in combination. This has the advantage of greatly improving the sensitivity when analyzing small amounts or very low concentrations of the sample.

DETAILED DESCRIPTION Hereinafter, the present invention will be described in more detail, and the facts apparent to those skilled in the art will be briefly mentioned or omitted in order not to obscure the subject matter of the present invention.

First, a brief description of Single drop microextraction (SDME). First, a method of forming a single drop will be described. Using the difference in pressure applied to both ends of the capillary tube, a few nanoliters of organic solvent is injected into the capillary tube, and the organic solvent and the aqueous solution in the capillary tube are ejected out of the capillary tube by applying pressure to the opposite side, and the tip is coated with an organic solvent. This creates a droplet with an aqueous acceptor phase. The droplets are extracted by an aqueous acceptor phase, concentrated, and injected into capillaries for analysis. This process is described in FIG.

2 is a view for explaining the principle of extraction. A single drop of an aqueous acceptor phase coated with a thin layer of organic phase can form at the end of the capillary. The analyte is concentrated into droplets by the pH difference between the aqueous acceptor phase and the aqueous donor phase. The microdroplets prepared by this method are very effective in obtaining the volume ratio of the largest aqueous solution among the conventional extraction methods. In addition, since the sample moves through the process of distributing to the organic layer, inorganic salts that do not melt into the organic layer do not move to the water-soluble phase, and thus may serve to remove inorganic salts that interfere with capillary electrophoresis. The problem is that the formation of a single drop is not easy or stable, and another problem is that the drop size is not large for experiments that require a larger amount of injection.

FIG. 3 shows that the drop formation for single drop microextraction (SDME) can be configured to be more stable and larger. The capillary surface has a weak interaction with organic solvents, and microdroplets have no physical support, which makes them very unstable when suspended in capillaries, making it difficult to apply agitation or stirring. The present invention simply solves this problem by inserting a teflon tube at the end of the capillary. Because of the affinity between the Teflon tube and the organic solvent (such as octanol), the support is reliably stabilized and the droplets are thicker than the capillary tube, making larger droplets possible. These large droplets are well suited for experiments that require large amounts of injection and provide the foundation for broadening the application of the SDME method. The size of the capillaries in the left and right figures of the drawings is the same. In the figure on the right, the Teflon tube is inserted into the capillary. However, as a result, the size of the single droplet in the right picture is quite large, and it can be confirmed that it is supported by the Teflon tube and formed stably. Without being limited to the Teflon tube, hydrophobic tubes such as PEEK tube can also be used.

According to the present invention, the concentration efficiency is very high when the extract is formed by forming a more stable and large single droplet. The present invention does not stop here but combines it with a sweeping method to provide a more efficient double concentration of sample. This will be referred to as a single drop microsweeping method. Tubes are added to increase the volume of droplets in single droplet microextraction for large volume sample injection for the sweeping mode. The tube may be a Teflon tube such as PTFE, PFA, FEP, PEEK tube, or the like. Figure 4 shows a schematic of the single droplet micro-sweeping method (SDME-sweeping method). Samples pre-concentrated in a large volume of single drop are sent through a capillary, which is then concentrated again in the stacking zone.

The sweeping process is shown in more detail in FIG. 5. A concentrated sample is extracted into a large volume single drop and injected into the capillary (a). After single drop microextraction, the samples are each separated by micellar electrokinetic chromatography (MEKC) with a sweeping effect. Sweeping in micelle electrodynamic chromatography can be defined as analytes being collected and accumulated by pseudostationary phases (micelles) that penetrate into the sample region under voltage. When there are micelle-structured cations in the buffer and analytes are anions in the sample, applying a voltage as shown in (b) causes the micelle-structured cations to temporarily form a stationary phase and the analytes accumulate in front of them. . And to detect these concentrated analytes. The programming may be performed by selecting an appropriate buffer according to the type of sample, adjusting the size of the EOF, and such a part may be easily understood by those skilled in the art, and a detailed description thereof will be omitted.

Even with small samples or extremely fine samples, the sensitivity is very good when using this double concentration method. This should be preceded by a single droplet microextraction stably and sufficiently large. The combination of large single-drop microextraction and the sweeping method provides a 10,000-fold improvement in sensitivity compared to normal capillary zone electrophoresis mode.

Hereinafter, the present invention will be described through an example in which a combination of SDME and sweeping is concentrated to concentrate fluorescein.

Comparison of single drop and single drop microextraction ( SDME )

A 60 cm fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a 50 μm inner diameter was fitted with a 0.3 mm, 1 mm outer diameter Teflon tube and filled with buffer. Then, the inlet of the capillary was placed in 1-octanol (Sigma, St. Louis, Mo.), and the outlet was placed in a buffer container. Octanol was injected into the capillary by raising the inlet end 9 cm for 3 minutes. The capillary was transferred to a cuvette containing an aqueous sample solution (primary phase). The cuvette was lowered 9 cm for 4 minutes to form droplets of buffered solution (octopus) covered with octanol at the capillary end. At this time, the buffer solution was prepared in pH 9.0 by adjusting the pH of 20 mM sodium phosphate (Sigma, St. Louis, MO) with 0.1 M HCl. A 1.5 mL aqueous sample solution of fluorescein (Sigma, St. Louis, MO) (primary phase) was prepared by adding 1490 μl of 0.10 M HCl (about pH 1) to 10 μl of standard solution of buffer. .

The difference between the use of Teflon tubes and the results in other cases was notable. This result is shown in FIG.

SDME - Sweeping  Through concentration

Fill the capillary with 10 mM phosphate buffer containing 20 mM dodecyltrimethylammonium bromide (DTAB), a cationic micelle, and 15% acetonitrile, and then inject 20 mM phosphate buffer without DTAB into the capillary for 3 min. . After injecting octanol for 3 minutes at a height of 9 cm, the injection portion of the capillary was immersed in the sample solution, and the solution was made 9 cm lower than the distal end for 4 minutes to make microdrops. After adjusting the height of the sample solution and the buffer solution at the distal end, the sample was extracted for 10 minutes, and an aqueous solution of microdrops was injected into the capillary tube for 2 minutes with a height difference of 10 cm. The capillary infusion was placed in a phosphate buffer solution containing DTAB and swept by adding 10 kV.

Comparative example

With the same sample, normal capillary zone electrophoresis, single droplet microextraction (SDME), single drop microextraction with large volumes using Teflon tubes as in the present invention (Large Volume SDME), in the case of simple sweeping, only the concentration method is used for the case of using the double concentration method (SDME-Sweeping) which performs single drop microextraction and sweeping using the Teflon tube of the present invention. Differently, the experiment was conducted under the same conditions as in the above example. The results are shown in FIG.

Compared to conventional capillary electrophoresis (CZE), there was a 100-fold improvement in sensitivity when microdroplets without Teflon tubes were extracted. By the way, when the Teflon tube is used to increase the microdroplets and increase the injection amount, the detection amount increases but the sensitivity does not increase in the large volume SDME of FIG. 6. In order to solve this problem, it was confirmed that the sweeping of FIG. Combining the two methods in the SDME-Sweeping of Figure 6 it was possible to improve a large sensitivity.

Compared to the usual capillary zone electrophoresis, when using a large volume single droplet microextraction (Large Volume SDME) using the Teflon tube as in the present invention, the sensitivity is increased by about 110 times. In the case of using the single drop microsweeping method (SDME-sweeping method) by forming a large drop as in the present invention, the sensitivity improved by approximately 10,000 times.

The present invention is not limited by the above-described embodiments of the present invention, and various changes can be added and changed within the scope of the technical spirit of the present invention from the viewpoint of those skilled in the art, and other equivalent embodiments will be possible. It is within the technical idea of this invention.

1 is a diagram illustrating a process of forming a single drop and extracting and injecting a sample.

2 is a view for explaining the principle of the SDME.

3 is a view showing the effect of forming a single drop by inserting the Teflon tube to the end of the capillary tube according to an embodiment of the present invention.

Figure 4 shows a schematic of the single droplet micro-sweeping method (SDME-sweeping method).

5 illustrates the sweeping process in more detail.

6 shows the results of improved assay sensitivity according to the enrichment effect of the present invention.

Claims (8)

In capillary electrophoresis, a single droplet is formed by forming a single drop at the end of the capillary tube for preconcentration of the sample, and a hydrophobic tube is placed at the end of the capillary tube so that a single drop can be stably formed. Characterized in that the fitting. The method of claim 1, The tubes include polytetrafluoroethylene (PTFE), perfluoroalkoxy polymer resin (PFA), fluorinated ethylene-propylene (FEP) and polyetheretherketones; PEEK) is selected from the group consisting of tubes. Polytetrafluoroethylene (PTFE) and perfluoroalkoxy polymers in capillaries for single drop microextraction by forming a single drop at the end of the capillary for preconcentration of the sample in capillary electrophoresis. Perfluoroalkoxy polymer resin (PFA), fluorinated ethylene-propylene (FEP) and polyetheretherketones (PEEK) are selected from the group consisting of tubes and the end capillary tube Capillary tube in losing form. A method for preconcentration of a sample in capillary electrophoresis, wherein the sample concentrated by the single drop microextraction method is again concentrated using a sweeping method (single drop microsweeping method; SDME-sweeping method). Preconcentration method of the sample, characterized in that the process. The method of claim 4, wherein the single drop micro-extraction method is polytetrafluoroethylene (PTFE), perfluoroalkoxy polymer resin (PFA), fluorinated ethylene-propylene; FEP) and polyetheretherketones (PEEK) material selected from the group consisting of a tube made of a pre-concentration method of the sample, characterized in that carried out by forming a large volume single drop inserted into the end of the capillary. The method according to claim 4 or 5, The sweeping method is characterized in that after single droplet microextraction, the analytes are collected and accumulated in a pseudostationary phase (micelles) by a micellar electrokinetic chromatography (MEKC) method having a sweeping effect. Preconcentration method of the sample. Forming a single drop at the capillary end of the capillary electrophoresis apparatus; Concentrating the sample in the single drop; Injecting the sample concentrated in a single drop into a capillary; Sweeping the sample injected into the capillary; preconcentration of the sample in capillary electrophoresis. The method of claim 7, wherein The single drop of capillary ends is polytetrafluoroethylene (PTFE), perfluoroalkoxy polymer resin (PFA), fluorinated ethylene-propylene (FEP) and polyetherether Selecting one from the group consisting of tubes of polyetheretherketones (PEEK) material, and inserting at the end of the capillary, the method comprising the step of forming a single droplet stably.

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