KR100858596B1 - Surface Treatment Method of Capillary for Droplet Formation and Surface treated Capillary, and Prouction Method of Droplet for Microextraction of Chemical Using Thereof - Google Patents

Abstract

The present invention relates to a capillary surface treatment method for forming microdroplets and a surface-treated capillary tube, and to a method for producing microdroplets for extracting the compound using the same, more specifically, the finely coated with an organic solvent at the end of the capillary tube In the process of concentrating a sample by making a drop, the method of improving the mutual affinity between the organic solvent and the capillary through surface treatment at the end of the capillary tube, and thereby allowing the microdrops to be stably formed. The present invention provides a capillary surface treatment method for improving the formation efficiency when forming fine droplets at the end of the capillary for micro-extraction, by applying a hydrophobic compound to the end of the capillary to give a hydrophobicity to the surface of the capillary In addition, it provides a capillary tube having such surface treatment characteristics. The present invention allows the microdrops to be formed very stably at the end of the capillary tube, the microdroplets for the micro-extraction made through this can be used very effectively for component analysis, such as capillary electrophoresis.

Capillary electrophoresis, capillary, hydrophobic, hydrophilic

Description

Surface treatment method of capillary for droplet formation and surface treated capillary, and prouction method of droplet for microextraction of Chemical Using Thereof}

1 is a schematic diagram illustrating the basic principle of the liquid / liquid / liquid extraction process illustrating the sample concentration process by the microdrops prepared by the present invention,

2 is a schematic view showing a state where the surface of the capillary tube is treated according to the present invention,

Figure 3 is a schematic diagram showing the mechanism for the microdrop production process and LLLE / CE process of the present invention,

Figure 4 is a schematic diagram showing the concentration of the sample by the liquid / liquid / liquid extraction process by microdrops,

5 is an enlarged photograph of microdrops formed according to the present invention;

FIG. 6 illustrates the principle of sample concentration by microdroplets in which hydrophilic materials such as small inorganic ions do not pass through an organic solvent layer, and only neutral specific substances pass through an organic solvent layer in liquid / liquid / liquid extraction. A schematic illustrating that the sample can be purified as well as concentrated.

The present invention relates to a surface treatment method of a capillary tube for forming microdroplets and a method for producing a microdroplet for surface extraction of the capillary tube, and to extract the compound using the same, more specifically, the fine coated with an organic solvent at the end of the capillary tube In the process of concentrating a sample by making a drop, the method of improving the mutual affinity between the organic solvent and the capillary through surface treatment at the end of the capillary tube, and thereby allowing the microdrops to be stably formed.

Methods such as gas chromatograpy and capilliary electrophoresis are widely used to analyze trace components. However, not only when the sample itself is a trace, but also trace components below picomol having a very low concentration in the sample require concentration before analysis.

In order to overcome the low concentration sensitivity of capillary electrophoresis (CE) among these concentration methods, sample stacking (Burgi, DS and Chien, RL, Anal. Chem. 1991 , 63 , 2042-2047), Intestinal-amplified sample injection (Chien, RL and Burgi, DS, J. Chromatogr. 1991 , 559 , 141-152; Chien, RL and Burgi, DS, J. Chromatogr. 1991 , 559 , 153-161) (He, Y. and Lee, HK, Anal. Chem. 1999 , 71 , 995-1001; Lee, JH et. Al., Electrophoresis 2000 , 21 , 930-934), transient isotachophoresis (Foret , F. et.al., J. Chromatogr. 1992 , 608 , 3-12; Foret, F. et.al., Electrophoresis 1993 , 14 , 417-428) and sweeping (Quirino, JP and Terabe, S., Science 1998 , 282 , 465-468; on-column concentration methods such as Quirino, JP and Terabe, S., Anal. Chem. 1999 , 71 , 1638-1644) are widely used. 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 associated with the CE can be used. Although solid phase extraction showed a thickening effect, it had the disadvantage of requiring desorption and poor reproducibility of concentration (Beattie, JH et. Al., Electrophoresis 1995 , 16 , 322-328; Strausbauch, MA et. Al., J Chromatogr., A 1995 , 717 , 279-291). As a result, liquid-liquid extraction (LLE) resulted in GC (Louter, AJH et. Al., J. Chromatogr., A 1999 , 842 , 391-426; Vreuls, JJ et. Al., J. Chromatogr., A 1999 , 856 , 279-314), LC (Jones, HK et. Al., J. Pharm. Biomed.Anal . 2002 , 29 , 221-228) and CE (Pedersen-Bjergaard, S. et. al., J. Chromatogr., A 2000 , 902 , 91-105). Various liquid-phase microextractions (LPME), which consumed less solvent while achieving higher concentrations than LLE, were performed by LC (Ma, MH and Cantwell, FF, Anal. Chem. 1998 , 70 , 3912-3919; Ma, MH and Cantwell, FF, Anal.Chem . 1999 , 71 , 388-393; Rasmussen, KE et. Al., J. Chromatogr., A 2000 , 873 , 3-11; Zhu, LY et. Al., J. Chromatogr., A 2001 , 924 , 407-414; Zhu, LY et. Al., J. Chromatogr., A 2002 , 963 , 231-237; Zhao, L. et. Al., J. Chromatogr., A 2002 , 963 , 239-248) and CE (Rasmussen, KE et. Al., J. Chromatogr., A 2000 , 873 , 3-11; Pedersen-Bjergaard, S. and Rasmussen, KE, Anal.Chem . 1999 , 71 , 2650-2656; Pedersen-Bjergaard, S. and Rasmussen, KE, Electrophoresis 2000 , 21 , 579-585; Halvorsen, TG et. Al., J. Chromatogr., A 2001 , 909 , 87-93; Halvorsen , TG et.al., J. Sep.Sci . 2001 , 24 , 615-622; Zhu, LY et.al., Anal.Chem . 2001 , 73 , 5655-5660; Pedersen-Bjergaard, S. et.al. ., J. Sep. Sci. 2002 , 25 , 141-146; Ho, TS et. Al., J. Ch romatogr., A 2002 , 963 , 3-17; Andersen, S. et.al., J. Chromatogr., A 2002 , 963 , 303-312). However, experimental manipulation was very complex and exhibited a memory effect (Pa'lmarsdo'ttir, S. et. Al., Anal. Chem . 1997 , 69 , 1732-1737; Thordarson, E. et. Al. ., Anal. Chem. 1996, 68, 2559-2563). Moreover, sensitivity improvement was insufficient.

On the other hand, in capillary electrophoresis, the method of concentrating a sample by making microdrops coated with an organic solvent is expected to increase detection sensitivity, but in reality, microdrops are not well formed, and most of the formed microdroplets The droplets actually formed due to the organic layer become a single phase, making it difficult to achieve the intended concentration, making it difficult to stabilize the formed droplets on the capillary end surface, and sufficient support force between the organic solvent and the capillary tube is sufficient. There is a problem that can not maintain the fine droplets. In addition, the concentration efficiency is affected by various factors such as significant time, temperature, partition coefficient, etc., and there is a problem in that it has to be adjusted, and there is a problem in that the stability of the microdrops is greatly reduced in the process of controlling the concentration. The above problems are substantially because the droplets in which the liquid / liquid / liquid layer is present do not form or are not made stable.

The present invention has been made to solve the above problems, and provides a method for treating the surface of the capillary tube to stably form the microdrops in which the liquid / liquid / liquid layer is present, and also for the fine extraction of the compound using the same It is to provide a method for producing microdroplets.

In order to achieve the above object, the present invention in the capillary surface treatment method for improving the formation efficiency when forming microdrops at the end of the capillary for fine extraction, by coating a hydrophobic compound on the end of the capillary Characterized by imparting hydrophobicity to the surface of the capillary, the hydrophobic compound may be selected from the group consisting of alkyltrimethoxysilane and PDMS, preferably octadecyltrimethoxysilane and in this case iii) acetone Washing the capillary surface; Ii) hydrolyzing the hydrophobic compound with ethanol as a solvent to form a coating solution; Iii) dipping the capillary end into the coating solution; Iii) surface treatment of the capillary tube, including the step of covalently bonding the silane to the capillary tube by curing and baking the capillary tube. In addition, in the capillary surface treated to improve the formation efficiency when forming fine droplets at the end of the capillary for fine extraction, characterized in that the end portion of the capillary tube is coated with a hydrophobic compound to have a hydrophobicity on the surface of the capillary Providing a surface treated capillary, i) coating the surface of the end of the capillary with a hydrophobic material; Ii) injecting the capillary filled with buffer into a hydrophobic organic solvent and injecting the organic solvent at the end of the capillary; Iii) putting the capillary terminal into which the hydrophobic organic solvent is injected into the sample solution to be concentrated; Iii) increasing the pressure on the opposite side of the capillary end to form microdroplets in the form of microdroplets in the form of the organic solvent surrounding the buffer solution, preparing microdroplets for the three-layer microextraction of liquid / liquid / liquid In the present method, the hydrophobic organic solvent may be selected from the group consisting of octanol, pentanol, octane, hexyl ether, sesame oil and peppermint oil.

Hereinafter, the present invention will be described in more detail.

The method for preparing microdroplets for three-layer microextraction of liquid / liquid / liquid is for liquid-liquid-liquid extraction (LLLE). Liquid-liquid-liquid extraction involves placing an organic solvent layer between the aqueous and aqueous layers and adjusting the pH of the two aqueous layers to adjust the sample material from the first aqueous layer (the aqueous donor layer) to the second aqueous layer (the aqueous phase). How to move. At this time, if the assumption that the distribution coefficient between the aqueous layer and the organic layer is large and the volume of the organic layer is established, the degree of concentration of the sample from the receiving layer to the receiving layer becomes the ratio of the volume of the two layers. And since the sample moves through the organic layer, the amount of the organic layer affects the extraction speed. Therefore, it is necessary to reduce the volume of the aqueous solution receiving layer and the organic layer in order to increase the extraction efficiency.

The present invention provides a method for producing nano-liter droplets at the end of the capillary by reducing the volume of the solution as described above. 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 coated with an organic solvent at the end. It will make a drop in the receiving layer. The microdroplets produced by the method of the present invention can obtain a volume ratio of the largest aqueous solution of the conventional extraction method, the extraction efficiency can be greatly increased. In addition, such microdroplets can be manufactured in an automated machine (eg, capillary can automatically adjust the height of the containment vessel over time), so the possibility of commercialization is high.

In the liquid-liquid-liquid extraction method, the sample moves through the distribution process to the organic layer, so that inorganic salts that do not dissolve in the organic layer do not migrate to the receiving layer. Using the above principle can also remove the inorganic salts that interfere with the analysis dissolved in the sample. In capillary electrophoresis, the presence of large amounts of inorganic salts in the sample often interferes with the analysis. Liquid-liquid-liquid extraction can provide a solution to this problem.

First, the basic principle of the liquid / liquid / liquid extraction process will be described with reference to FIG.

LPME consists of two consecutive reversible extractions. The analyte in the water-soluble main phase (a1) is first extracted into the organic phase (o) and then back extracted into the water-soluble receiving phase (a2). In equilibrium, the enrichment factor (EF), defined as the ratio of the equilibrium concentration ( C a2, eq) in the receiving phase to the initial concentration ( C a1, i) of the analyte in the primary phase, can be expressed as Ma, MH and Cantwell, FF, Anal. Chem. 1998 , 70 , 3912-3919).

Where V represents the volume of the phase indicated by subscripts, and the distribution coefficients of D1 and D2 are defined as follows.

And,

Assume monohydrogen production analyte HA with pKa. Assuming that only the neutral form is soluble in the receiving phase, the distribution coefficient D can be given as

Where K is the partition coefficient of the neutral mold.

In equation 1, the ratio of the partition coefficients is

For three hydrogen analytes H ₃ A ⁺ , such as fluorescein and FITC, the formula for the ratio of the distribution coefficient is as follows.

For pHa1 = 1.0 and pHa2 = 9.5, the distribution coefficient ratio for fluorescein using p K a1 = 2.13, p K a2 = 4.44 and p K a3 = 6.36 for fluorescein was D 2 / D 1 = 9.1 X 10 ^-8 (Diehl, H. and Markuszewski, R., Talanta 1985 , 32 , 159-165). Then, at a typical volume of V a1 = 1 mL, V o is about 5 nl and V a2 = 100 nl, the concentration ratio is approximated as follows.

Because the effect of concentration is the difference between the volume of the water-soluble main phase (a1) and the water-soluble receiving phase (a2), it is very important to reduce the volume of the aqueous phase of the aqueous phase, and thus to create a nanoliter droplet at the end of the capillary. It can be very groundbreaking. In addition, since the sample moves through the organic layer, the amount and thickness of the organic layer affect the extraction speed. In the present invention, droplets of the organic layer are formed at the end of the capillary tube. The water-soluble receiving phase is located inside the organic layer, and the water-soluble main phase is located outside the organic layer, thereby forming the micro-droplets by surrounding the water-soluble receiving phase and forming a microdrop. To achieve.

However, when the droplet is unstable, it will be very difficult to reduce the thickness and volume of the organic layer, so the stable formation of micro droplets will be a very important problem. On the other hand, it is clear that basic analytes can be easily achieved by reversing the pH of the main phase and the receiving phase.

In the present invention, the surface of the capillary tube is treated to form stable microdrops, and FIG. 2 shows a schematic view of the capillary tube with the surface treated.

Hydrophilic capillary surfaces have poor interaction with organic solvents, and microdroplets lack physical support, making them very unstable when suspended in capillaries, making it difficult to apply agitation or stirring. There is also difficulty in generating microdrops due to excessive polyimide coating.

If the interaction between the organic solvent and the capillary is weak, and the support force is not sufficient, the microdroplets cannot be maintained and the capillary surface treatment is important for maintaining the production probability and stability of the microdroplets. As can be seen, coating with a hydrophobic material solved the above problem.

The hydrophobic material may be alkyl trimethoxysilane (R-trimethoxysilane), polydimethylsiloxane (PDMS), or the like. Preferably, alkyl trimethoxysilane having 12 or more carbon atoms is used to have sufficient hydrophobicity, more preferably octa. Decyltrimethoxysilane is used.

In the case of using silane, generally, iii) washing the capillary surface with acetone; Ii) hydrolyzing the hydrophobic compound with ethanol as a solvent to form a coating solution; Iii) dipping the capillary end into the coating solution; V) curing and baking the capillary to covalently bond the silane to the capillary; It will include.

Example 1 Surface Treatment of Capillary Tubes

First, the capillary was immersed in acetone for 3 minutes to keep the capillary inside filled with a hydrophilic buffer solution to prevent washing and excessive coating of the capillary surface. Using ethanol as a solvent, 5% octadecyltrimethoxysilane, 0.1% acetic acid solution of the following structural formula was prepared, and was allowed to hydrolyze for at least 5 minutes. The capillary tip was added to the silane solution for a short time of about 1 second and then removed, and then dried at room temperature in air for 1 minute and 30 seconds to form a covalent bond with the capillary surface. As a result, the silica surface of the capillary became hydrophobic and the affinity with octanol used as an organic solvent in the preparation of microdrops was increased.

Meanwhile, in the case of PDMS, a method of forming a covalent bond with the capillary surface by slightly dipping it as it is and then drying it may be used, and there may be a slight change in the coating method depending on the type of the hydrophobic material. Is to make the surface of the capillary hydrophobic to strengthen the affinity with the organic layer.

Schematic diagram showing the mechanism for the microdrop production process and LLLE / CE process of the present invention in conjunction with capillary action using a hydrophobic surface-treated capillary is shown in FIG. The organic solvent used here may be selected according to the characteristics of the sample to be concentrated, and should not be easily mixed with water, for example, octanol, pentanol, octane, hexylether Sesame oil or peppermint oil is also possible. In the case of an alcohol having a carbon number of pentanol or more, octanol which can be used because it is not easily mixed with water and can exhibit sufficient hydrophobicity is more preferable.

Find out how to form microdrops. In this example a surface treated capillary was used. First, octanol is injected into the capillary filled with an aqueous solution. As shown in the figure, the end of the capillary tube is put in a container containing octanol, and pressure is applied so that the octanol enters the capillary tube. The next step is to form a drop, where the capillary is placed in the sample solution to be extracted and pressurized from the opposite side (indicated by lifting the opposite buffer container) to give octanol and then to the aqueous solution in the capillary. do. At this time, since octanol has a large affinity with the capillary surface, microdroplets as shown in FIG. 5 are prepared. The sample solution is considerably larger in volume than the microdroplets and corresponds to a water-soluble main phase, and the aqueous solution layer surrounded by octanol corresponds to a water-soluble receiving phase. In addition, the octanol layer, which is an organic layer, is hydrophobicly surface-treated at the end of the capillary tube, and thus has a high affinity, thereby achieving stable microdroplets and having a very thin thickness. The pH of the main phase is 1.0, the pH of the receiving phase is 9.5, and the liquid / liquid / liquid extraction mentioned above is achieved, and when the concentration is sufficiently high enough to be analyzed, it is analyzed in connection with capillary electrophoresis. This approach consumes very little solvent and no special additional equipment is required. The method for producing a microdroplet is iii) coating the surface of the end of the capillary with a hydrophobic material; Ii) injecting the capillary filled with buffer into a hydrophobic organic solvent and injecting the organic solvent at the end of the capillary; Iii) putting the capillary terminal into which the hydrophobic organic solvent is injected into the sample solution to be concentrated; Iii) increasing the pressure on the opposite side of the capillary end to form microdroplets in the form of microdroplets in the form of the organic solvent surrounding the buffer solution, preparing microdroplets for the three-layer microextraction of liquid / liquid / liquid It can be seen as a way.

Figure 4 is a schematic diagram showing the concentration of the sample by the liquid / liquid / liquid extraction process by the microdrops and specific examples are as follows.

Example 2 Formation of Microdroplets

A 50 cm fused silica capillary (Polymicro Technologies, Phoenix, AZ) with a 75 μm inner diameter was used and the capillary was 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. The octopus was injected into the capillary by raising the end of the injection section by 12 cm, and the injection amount was controlled by the injection time. The capillary was transferred to capillary tongs on a microcentrifuge tube containing 1.0 mL of aqueous sample solution (primary phase). The microcentrifuge tube was lowered by 13 cm for 30 seconds to form droplets of buffered solution (octopus) covered with octanol at the capillary end. At this time, the buffer solution was prepared to pH 9.5 by adjusting the pH of 20 mM sodium borate (Sigma, St. Louis, MO) with 0.1 M NaOH. A 1 ml aqueous sample solution of fluorescein (Sigma, St. Louis, MO) and fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO) (primary phase) is 10 μl of a standard solution of buffer. To 990 μl of 0.10 M HCl (about pH 1) was added.

FIG. 6 illustrates the principle of sample concentration by microdroplets in which hydrophilic materials such as small inorganic ions do not pass through an organic solvent layer, and only neutral specific substances pass through an organic solvent layer in liquid / liquid / liquid extraction. It indicates that the sample can be purified as well as concentrated. In general, high salt concentrations in CE are responsible for destacking and low efficiency separation. Even when the main phase contains 0.1 M NaCl in addition to 0.1 M HCl, sufficient concentration can be achieved without reducing the peak efficiency or the concentration ratio, which means that the method of the present invention is a physiological sample, such as a cell saline with a high saline concentration. It can also be applied to.

The above embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the embodiments.

As described above, the present invention enables the microdroplets to be formed very stably at the end of the capillary tube, through which the microdroplets can be used very effectively for component analysis, such as capillary electrophoresis.

Claims (9)

delete delete delete delete delete delete delete Iii) coating the surface of the end of the capillary with a hydrophobic material; Ii) injecting the capillary filled with buffer into a hydrophobic organic solvent and injecting the organic solvent at the end of the capillary; Iii) putting the capillary terminal into which the hydrophobic organic solvent is injected into the sample solution to be concentrated; Iii) increasing the pressure on the opposite side of the capillary end to form microdroplets in which the organic solvent surrounds the buffer solution to form microdroplets in the form of liquid / liquid / liquid. Way. The method of claim 8, wherein the hydrophobic organic solvent is selected from the group consisting of octanol, pentanol, octane, hexyl ether, sesame oil and peppermint oil.

Images