CHOI, Ki hwan (Hanareum mauel 501ho, Shinlim bondong Kwanak-gu, Seoul 151-029, KR)
[CLAIMS] [Claim 1] <66> A method for surface tailoring of a capillary to to increase the efficiency of droplet formation in microextraction,
<67> wherein the tip of capillary is coated with hydrophobic compound and therefore has hydrophobic property.
[Claim 2]
<68> The method of claim 1, wherein said the hydrophobic compound is seleted among the group of alkyltrimethoxysilane and PDMS.
[Claim 3]
<69> The method of claim 1, wherein said hydrophobic compound is octadecyltrimethoxysi lane.
[Claim 4]
<70> The method of claim 3, wherein the surface tailoring procedure includes the next step, 1) washing of surface using acetone; 2) preparation of coating solution by hydrolysis of hydrophobic compounds in ethanol solvent; 3) dipping of capillary tip to the coating solution; 4) surface curing and formation of covalent bond. [Claim 5]
<7i> A surface tailored capillary which is coated with hydrophobic compound on the tip to increase the efficiency of droplet formation in microextraction.
[Claim 6]
<72> The capillary of claim 5, wherein said the hydrophobic compound is seleted among the group of alkyltrimethoxysilane and PDMS.
[Claim 7]
<73> The capillary of claim 5, wherein said hydrophobic compound is octadecyltrimethoxysi lane.
[Claim 8]
<74> A method for droplet formation in liquid-liquid-liquid extraction including the step of: 1) hydrophobic coating of capillary tip surface; 2) injection of organic solvent to the capillary filled with buffer solution; 3) positioning of capillary to the sample solution; 4) formation of droplet by applying pressure to the opposite side of the capillary.
[Claim 9]
<75> The method of claim 8, wherein said hydrophobic organic solvent is seleted among the group of octanol, pentanol, octane, hexylether, sesame oil and peppermint oil. |
[DESCRIPTION] [Invention Title]
SURFACE TREATMENT METHOD OF CAPILLARY FOR DROPLET FORMATION AND SURFACE TREATED CAPILLARY, AND PRODUCTION METHOD OF DROPLET FOR MICROEXTRACTION OF CHEMICAL USING THEREOF [Technical Field]
<i> This present invention relates to surface tailoring method of capillary for droplet, surface-modified capillary, and fabrication method of droplet for microextraction. More particularly the present invention provides a stabilization method of the aqueous droplet coated with organic solvent on the tip of a capillary by the increase of the affinity between the organic solvent and surface of the capillary in microextraction. [Background Art]
<2> Capillary electrophoresis is used as a versatile separation technique in various fields. However, poor absorbance detection sensitivity prohibits its application to trace analysis. Several on-column concentration techniques has been developed to overcome the low sensitivity, including sample stacking (Burgi, D.S. and Chien, R.L., Anal. Chem. 1991, 63, 2042-2047), field- amplified sample injection (Chien, R.L. and Burgi, D.S., J. Chromatogr. 1991, 559, 141-152; Chien, R.L. and Burgi, D.S., J. Chromatogr. 1991, 559, 153- 161), large volume sample stacking (He, Y. and Lee, H.K., Anal. Chem. 1999, 71, 995-1001; Lee, J.H. 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), sweeping (Quirino, J.P. and Terabe, S., Science 1998, 282, 465-468; Quirino, J.P. and Terabe, S., Anal. Chem. 1999, 71, 1638-1644)
<3> The advantages of on-column concentration are that no mechanical modification of the column is required and the procedure is relatively simple with little manipulation of buffer and sample systems. However, its application is limited to cases satisfying specific requirements for the run buffer and sample solutions. As an alternative to these techniques, sample
preconcentration methods coupled with CE can be employed. Although solid- phase extraction, which was initially introduced to CE as a pretreatment technique, showed concentration effects, a solvent desorption step was required and sample recovery was not reproducible (Beattie, J.H. et . al . , Electrophoresis 1995, 16, 322-328; Strausbauch, M.A. et . al . , /. Chrowatogr. , A 1995, 717, 279-291). Liquid-liquid extraction (LLE) has been applied to GCCLouter, A.J.H. et . al . , /. Chromatogr. , A 1999, 842, 391-426; Vreuls, J.J. et. al., J. Chrowatogr., A 1999, 856, 279-314), LCUones, H.K. 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 microextraction (LPME) schemes achieving higher concentration effects and consuming much less solvent than LLE were applied to LC(Ma, M.H. and Cantwell, F.F., Anal. Chew. 1998, 70, 3912-3919; Ma, M.H. andCantwell, F.F., Anal. Chew. 1999, 71, 388-393; Rasmussen, K.E. et . al . , J. Chrowatogr., A
2000, 873, 3-11; Zhu, L.Y. et . al . , J. Chromatogr., A 2001, 924, 407-414; Zhu, L.Y. et. al., J. Chrowatogr., A 2002, 963, 231-237; Zhao, L. et . al . , /. Chrowatogr., A 2002, 963, 239-248) and CE(Rasmussen, K.E. et . al . , /. Chrowatogr. , A 2000, 873, 3-11; Pedersen-Bjergaard, S. and Rasmussen, K.E., Anal. Chew. 1999, 71, 2650-2656; Pedersen-Bjergaard, S. and Rasmussen, K.E. , Electrophoresis 2000, 21, 579-585; Halvorsen, T.G. et . al . , J. Chromatogr., A
2001, 909, 87-93; Halvorsen, T.G. et . al., /. Sep. Sci. 2001, 24, 615-622; Zhu, L.Y. et. al., Anal. Chem. 2001, 73, 5655-5660; Pedersen-Bjergaard, S. et. al., /. Sep. Sci. 2002, 25, 141-146; Ho, T.S. et . al . , /. Chromatogr., A
2002, 963, 3-17; Andersen, S. et . al . , /. Chromatogr., ,42002, 963, 303-312). However, the experimental arrangements were rather complicated and exhibited memory effects (Pa' lmarsdo'ttir , S. et . al., Anal. Chew. 1997, 69, 1732-1737; Thordarson, E. et . al., Anal. Chew. 1996, 68, 2559-2563). Moreover, the sensitivity improvement was insufficient.
[Disclosurel [Technical Problem] <4> In capillary electrophoresis, preconcentration method using droplet
coated with organic solvent is expected to increase the detection sensitivity. However, formation of droplet is difficult due to the leakage of droplet through organic solvent. And, decrease of droplet stability in vigorous stirring and long extraction is another problem. [Technical Solution]
<5> The invention, developed to solve above mentioned problems, provides the surface tailoring method to form stable aqueous droplet coated with organic solvent, and method to fabricate droplet for the microextraction.
<6> To achieve mentioned objectives, hydrophobic treatment of surface of the capillary tip is main feature of this method. Hydrophobic agents includes alkyltrimethoxysi lanes and PDMS, and octadecyltrimethoxysilane is desirable. Surface treatment procedures are as follows: 1) washing of surface using acetone; 2) preparation of coating solution by hydrolysis of hydrophobic compounds in ethanol solvent; 3) dipping of capillary tip to the coating solution; 4) surface curing and formation of covalent bond.
<7> Also, formation of droplet for the three-phase extraction (liquid- liquid-liquid extraction) is demonstrated as follows: 1) hydrophobic coating of capillary tip surface; 2) injection of organic solvent to the capillary filled with buffer solution; 3) positioning of capillary to the sample solution; 4) formation of droplet by applying pressure to the opposite side of the capillary. Organic solvent for the three-phase extraction can be choose from various solvent immisible to water such as octanol, pentanol, octane, hexylether, sesame oil, peppermint oil.
[Advantageous Effects]
<9> As shown in previous section, this invention suggest stable formation of droplet. As a result, droplet can be utilized effectively in capillary electrophoresis. [Description of Drawings]
<io> FIG. 1 is a schematic representation of principle of liquid-liquid- liquid extraction, explaining the concentration process.
<π> FIG. 2 is a schematic representation of tailored surface of a capillary of an this invention.
<i2> FIG. 3 is a schematic representation of droplet formation procedure of this invention.
<13> FIG. 4 is a schematic representation of preconcentration by liquid- liquid-liquid extraction.
<14> FIG. 5 is a picture of droplet according to this invention.
<15> FIG. 6 is a schematic representation of cleanup mechanism. Inorganic ions and hydrophilic compounds cannot penetrate the organic solvent and specific compounds can be preconcentrated. [Best Mode]
<16> Detailed description will be explained.
<17> Aqueous droplet coated with organic solvent is formed for the liquid- liquid-liquid extraction. Liquid-liquid-liquid extraction is preconcentration method using organic solvent as liquid solvent as a membrane between aqueous donor and aqueous acceptor phase. By manipulation of the pH of the two aqueous phase, analytes can be concentrated to the aqueous acceptor phase. If the distribution constant between aqueous acceptor phase and organic phase is very large and the volume of organic phase negligible compared to the volume of aqueous donor phase, the enrichment factor can be approximated as the ratio of the volume of two aqueous phases. Furthermore, amount of organic solvent affects the extraction speed since analytes are partitioned through the organic solvent. Therefore, reduction of volumes of organic phase and aqueous acceptor phase is essential for the efficient extraction.
<18>
<19> This invention provides the method to form nanoliter-sized droplet on the tip of the capillary. The droplet is made by the injection of nanoliter- sized organic solvent, and emission of organic solvent and aqueous solution by applying pressure to the opposite side of the capillary. Since the volume ratio of two aqueous solutions are largest among developed, extraction efficiency can be increased. Moreover, automation by a commercial instrument can increase the possibility of practical use.
<20>
<2i> Since organic solvent prevent partitioning of inorganic ions to aqueous acceptor phase, removal of inorganic ions hindering CE analysis can give solution.
<22>
<23> FIG 1. shows the basic principle of liquid-liquid-liquid extraction.
<24> SDME involves two consecutive extractions. The analytes in the aqueous donor phase (al) is extracted into organic solvent (o) and then back- extracted into the aqueous acceptor phase (a2). Enrichment factor, defined as ratio of the initial concentration in the donor phase and equilibrium concentration in the acceptor phase is expressed as follows (Ma, M.H. and Cantwell, F.F., Anal. Chem. 1998, 70, 3912-3919):
<25> (1)
EF = C *2. eg _ 1
<26> C -fl D 2 ZD 1 + D 2 (V 0 /Y 11 ) + V 12 ZV 11
<27> Where, Vs are the volume of each phases, and Dl and D2 are distribution constants defined by followings.
<28> (2)
<29> ** ■ •*
<30> and,
<31> (3)
<32>
<33> If K 2 is very small and V 0 < V a2 , concentration ratio of (1) is approximated to volume ratio of two layers. Let us assume VaI= 1 mL, Vo= 5 nL, Va2= 100 nL, EF has the following approximation.
<34> (4)
EF = -^i- =-> 10 4 <35>
<36> The affinity between sample extracted to acceptor phase and organic solvent needs reducing to make K2 very small, and the ajustment of pH is generally used. Extracted sample can not be distributed over organic layer in high pH of acceptor phase when extracting acidic material. In low pH of donor phase, distribution over organic layer would be produced well.
<37> It is crucial to reduce the volume of the aqueous acceptor phase for large enrichment factor, therefore, this method to make nano-liter sized droplet on the tip of capillary is innovative. Thin organic layer, also, increase the extraction kinetics. In this invention, liquid-liquid-liquid extraction(LLLE) is achieved by the formation of aqueous acceptor droplet coated with organic solvent in the aqueous donor phase.
<38> It is not easy to decrease the volume and thickness of organic solvent in unstable drop condition. For basic analytes, preconcentration using LLLE can be easily achieved by reversing the pH of the donor and acceptor phases.
<39>
<40> This invention relates to surface tailoring for the stable formation of droplet as shown in FIG. 2. <4i> Week intreaction between hydrophilic capillary surface and organic solvent, and absense of physical support make droplet unstable under vigorous agitation conditions. And excess coating of polyimide make it difficult to produce droplet.
<42>
<43> The importance of surface tailoring of capillary was confirmed due to the lack of support, and hydrophobic coating was introduced to overcome the drawback as shown in figure.
<44> Hydrophobic compounds such as Alkyltrimethoxysilane, polydimethylsiloxane can be used as surface tailoring agent. Alkyltrimethoxysi lanes having more 12 carbon are preferred. In preferred embodiments of this invention, octadecyltrimethoxysilane is used.
<45>
<46> In case of si lane is used as tailoring agent, treatment procedure includes 1) washing of surface using acetone; 2) preparation of coating solution by hydrolysis of hydrophobic compounds in ethanol solvent; 3) dipping of capillary tip to the coating solution; 4) surface curing and formation of covalent bond.
<47>
[Mode for Invention]
<48> <Imbodiment 1> Surface treatment of the capillary
<49> After being washed in ethanol for 3 min, the capillary inlet tip was immersed in a surface coating solution (5 vol % ODTS, 0.1 vol % acetic acid in ethanol) for 1 s then, in order to form covalent bonding, the inlet tip was dried in an empty vial for 1.5 min. Through this treatment, capillary surface became hydrophobic, thus increasing the affinity to octanol.
<50>
<5i> When PDMS was used, immersing and drying was sufficient for hydrophobic treatment. Although variation of coating methods may be possible, main feature of this invention is the increase of affinity of capillary surface to organic solvent by hydrophobic treatment.
<52>
<53> Droplet formation procedure in capillary electrophoresis was shown in FIG 3. Organic solvent, immiscible to water, can be selected according to the characteristics of analytes including octanol, pentanol, octane, hexylether, sesame oil, peppermint oil. Although alcohol having more than 5 carbon can be
O
used, octanol is preferred organic solvent.
<54>
<55> Let explain the drop formation method in detail. In above example, surface tailored capillary was used. Octanol was introduced to the capillary filled with aqueous acceptor phase. For the drop formation, the inlet end of the capillary was placed in the donor phase and backpressure was applied to the outlet vial. Then, aqueous droplet coated with octanol was formed as shown in FIG. 5. Volume of sample solution is much larger than that of droplet and aqueous solution inside of the droplet is aqueous acceptor phase. And octanol worked as organic solvent became to have strong affinity to the hydrophobic surface of the capillary and the thickness of organic solvent became very thin. The pH of the donor and acceptor phases were 1.0 and 9.5 respectively. The concentrated analytes in the acceptor phase was injected to the capillary and analyzed by capillary electrophoresis system. Consumption of toxic organic solvent was minimized and additional instruments were not required. As described above, this invention suggest the droplet formation method for microextraction, which consists of 1) hydrophobic coating of capillary tip surface! 2) injection of organic solvent to the capillary filled with buffer solution; 3) positioning of capillary to the sample solution! 4) formation of droplet by applying pressure to the opposite side of the capillary.
<56>
<57> FIG. 4 is a schematic representation of preconcentration by liquid- liquid-liquid extraction and one embodiment of this invention is described as follows:
<58> <Imbodiment 2> Formation of droplet
<59> 50-cm fused silica capillaryCPolymicro Technologies, Phoenix, AZ) of 75 [M inner diameter was filled with buffer solution. The inlet end of the capillary was then placed in 1-octanol and the outlet end in a run buffer reservoir. Octanol (Sigma, St. Louis, MO) was injected into the capillary by raising the inlet end by 12 cm, and the amount of injection was controlled by
the injection time. After the inlet end was wiped with a piece of lint-free tissue to remove octanol on the outside of the capillary, the capillary was transferred to a capillary holder on a microcentrifuge tube containing an aqueous sample solution of 1.0 mL (donor phase). The microcentrifuge tube was lowered by 13 cm for 30 s to form a drop of the run buffer solution (acceptor phase) covered with octanol at the capillary tip. A buffer solution was prepared by adjusting the pH of 20 mM sodium borate (Sigma, St. Louis, MO) with 0.1 M NaOH to pH 9.50. A 1-mL aqueous sample solution (donor phase) of fluorescein (Sigma, St. Louis, MO) and FITC (Sigma, St. Louis, MO) was prepared by adding 10 uL of the standard solution in the buffer to 990 μJL of 0.10 M HCl (pH ~1).
<60>
<6i> FIG 6. shows the cleanup mechanism. Inorganic ions and hydrophilic compounds cannot penetrate the organic solvent and specific compounds can be preconcentrated. High salt solutions usually cause destacking and poor separation efficiency in CE. Even when the donor phase contained 0.1 M NaCl in addition to 0.1 M HCl, the enrichment was accomplished without a decrease in peak efficiency or enrichment factor, implying that this method could be applied to physiological samples in highly saline matrixes.
<62>
<63> Mentioned imbodiment is only one example and this invention is not limited to this imbodiment.
<64>