WO2018117309A1

WO2018117309A1 - Capillary electrophoresis-electrospray ionization-mass spectrometry using single drop microextraction and large-volume stacking

Info

Publication number: WO2018117309A1
Application number: PCT/KR2016/015187
Authority: WO
Inventors: 최기환; 정두수; 김지혜
Original assignee: 한국표준과학연구원; 서울대학교산학협력단
Priority date: 2016-12-23
Filing date: 2016-12-23
Publication date: 2018-06-28
Also published as: KR102201770B1; KR20190095466A

Abstract

The present invention relates to a method for continuously stacking, separating, and analyzing a sample extracted as an organic droplet, through the linking of single drop microextraction (SDME) and large-volume stacking using an electroosmotic flow pump (LVSEP) with capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). By applying the method of the present invention, the concentration coefficient of an extracted sample can be remarkably improved, the sample can be analyzed with a high sensitivity, and a soil pollutant sample having a low concentration can be conveniently and usefully analyzed without passing through a complex process.

Description

Specification

Title of the Invention: Capillary Electrophoresis using Single Droplet Microscopy and Large-Boot Tacking Methods-Electrospray Ionization-Mass Spectrometry

[1] The present invention uses the single drop microscopy (SDME) and the large volume stacking method (LVSEP) using an electroosmotic flow pump (CE-ESI-MS) for capillary electrophoresis and electrospray.

Ionization-Mass Spectrometry) to continuously stack, separate and analyze samples extracted from organic droplets.

Background

[2] Capillary electrophoresis is an excellent method of separation of charges, but at very low concentrations, many efforts have been made to increase its sensitivity, among which there is a method of stacking samples in capillaries. Large-volume stacking using the electroosmotic flow pump (LVSEP) is a method in which a matrix of samples is subjected to electroosmotic flow after a large amount of negative ion sample dissolved in a solution with low electrical conductivity is injected into the capillary. The high concentration effect can be expected by the method that the sample is concentrated with high separation by being removed by the electroosmotic flow (EOF). LVSEP is a relatively easy method to expect a high concentration effect, and recently, it has been reported to use it in conjunction with MS (Korean Patent 10-1144974). In the reported method, the buffer vial containing the run buffer is reported. The buffer vial was placed in the capillary outlet and added while concentrated, but the pre-treatment of the sample was cumbersome, as the efficiency of stacking was proportional to the conductivity ratio between the matrix of the sample and the run buffer.

[3] Single drop microextraction (SDME)

Of microliter volume at the tip of the microsyringe needle

It is a method of making a single drop and extracting it using the droplet as an extractant. Based on this basic approach of the SDME,

Increasing the volume of the acceptor can be made larger by increasing the volume difference with the donor.

A method of performing pretreatment and separation analysis of samples at once by incorporating the MS method has been reported (Korean Patent No. 10-1160389).

[4] However, the previously reported individual LVSEPs and SDMEs can be used to analyze low-contaminant samples in soils with high concentration factors with high sensitivity.

Basically, it was observed to be unsatisfactory, and the analysis also required a complex process that required complex pretreatment and concentrated concentrations of sample material at low concentrations. There is a need for a high sensitivity analysis of the contained low concentration pollutant samples with high concentration coefficients without complex processes.

Detailed description of the invention

Technical task

[5] In the present invention, according to the above requirements, the Single Droplet Microscopy (SDME) and

All of the large piece piece stacking methods (LVSEP) using an electroosmotic flow pump

In conjunction with CE-ESI-MS (Capillary Electrophoresis-Electrospray Coagulation-Mass Spectrometry), we intend to provide a method for the continuous stacking, separation and analysis of samples extracted with an organic chamber.

[6] In the present invention, SDME and LVSEP are both linked to CE-ESI-MS to provide a method for significantly improving the sample concentration factor and analyzing the sample with high sensitivity.

[7] In the present invention, SDME and LVSEP are both linked to CE-ESI-MS to significantly improve the concentration coefficient of extracted low-contaminant soil contaminant samples without complex process, and to analyze samples with high sensitivity. Wish to provide.

Task solution

[8] In the following, the present invention will be described in more detail. In order to avoid obscuring the subject matter of the present invention, the facts obvious to the ordinary engineer are briefly mentioned or omitted. For example, a typical ESI-MS mass spectrometer is well known to ordinary technicians, and its structural details are omitted.

[9]

[10] For the above problem, the present invention

[1 1] a) filling the run buffer into the capillary;

[12] b) at the capillary outlet on the orifice side of the mass spectrometer (MS)

Placing an outlet storage container containing a run buffer to lock the capillary outlet;

[13] c) After injecting the organic acceptor pentanol into the capillary, remove the inlet portion of the capillary.

Dipping in donor solution (sample solution);

[14] d) drawing an organic droplet at the end of the inlet portion of the capillary by pulling the pentanol injected into the capillary toward the inlet portion of the capillary;

E) extracting a sample from the donor solution into the droplet;

[16] f) Injecting the acceptor solution into the capillary, in which the sample is concentrated,

Dipping the inlet portion into the run buffer;

[17] g) stacking the extracted sample by applying a reverse voltage to the inlet portion of the capillary and observing the current change of the capillary electrophoresis to remove the outlet storage container when the change of current occurs suddenly; and [18] h) removing the sheath liquid from the mass spectrometer and applying an electrospray ionization voltage (ESI voltage) to move the stacked concentrate sample to the mass spectrometer;

[19] characterized by the Single Drop Microanalysis (SDME) and

Continuously stacking, separating and analyzing samples extracted into organic chambers by effectively linking the large volume piece stacking method (LVSEP) using an electroosmotic flow pump with CE-ESI-MS (Capillary electrophoresis-electrospray ionization-mass spectrometry). To provide a way.

[20]

[21] In the method of the present invention, the organic droplets formed in step d)

In order to remain stable at the end of the inlet portion, the capillary inlet portion may be coated with a hydrophobic support, Teflon sleeve (FIG. 1), wherein the organic droplets are sufficient for subsequent application of LVSEP. It is desirable to have a size and that the organic inlet portion of the capillary tube is coated with a Teflon sleeve, which is a hydrophobic support, in order to ensure that the droplets are stable enough to facilitate the application of LVSEP.

[22] The capillary tube of the capillary electrophoresis system in step b) of the method of the present invention.

The outlet reservoir containing the buffer solution may be detachably mounted between the outlet and the sampler orifice cone of the mass spectrometer, with the capillary outlet configured to be submerged in contact with the buffer solution of the outlet reservoir. It is very important.

[23] In step c) of the method of the present invention, in order to facilitate the implementation of the methodological features of SDME and LVSEP interoperability, pentanol is adopted as an organic memory and an extraction solvent. When high-alcohol octanol is injected into the capillary to interlock SDME and LVSEP, due to the high viscosity of octanol (9.85 cP, 20 ^< C), the sample matrix of LVSEP application does not remove octanol from the capillary smoothly. In the step c) of the method of the present invention, a small amount of pentane is preferably injected into the capillary, more specifically, 5 to 20% of the total capillary volume. This is preferable (FIG. 2 (a)).

[24] In step c) of the method of the present invention, the sample applied to the donor solution

Preferably, it may be a soil contaminant.

^{^{(4-chloro-2-methylphenoxy)}} acetic acid ^{^{((4 -Chloro- 2 -methylphenoxy) acetic}} acid; MCPA: pK a 3A4), 2,4,5-trifluoroacetic acid (2,4,5-trifluorobenzoic acid: pK _a 2.53), 2,3,4,5-tetrafluorobenzoic acid (2,3,4,5-tetrafluorobenzoic acid: pK _a 2.Sl),

2-methyl-4,6-dinitrophenol (2-Methyl-4,6-dinitrophenol; 4,6-DNOC: pi ^ _a 4.70), 2,4-dinitrophenol (2,4-dinitrophenol; 2, 4-DNP: ΚΑ- Ί) or a combination thereof ᅳ The above exemplary pollutants are harmful substances that may be included in the soil, This also applies to useful analytes that may be applied to the method. When applying the exemplary contaminant to the donor solution, the pH of the donor solution is preferably between 0.5 and 2. The exemplary contaminant is between 2.53 and 4.70. As long as the pi _a range is indicated, the pH of the donor solution must be 0.5 to 2, so that the exemplary pollutant is present in the neutral state, and then, in step e), the exemplary pollutant in the neutral state is retained. It can be extracted smoothly by inpentanol, and the low electrical conductivity of the acceptor solution is also ensured, which in turn facilitates the application of LVSEP. On the other hand, in the presence of some of the exemplary contaminants in the donor solution, these forms, such as salts or basic compounds, are difficult to extract smoothly by the organic acceptor pentanol.Soil contaminants as the sample applied to the donor solution The solution may preferably contain low concentrations below 70 nM and more preferably between 1 and 70 nM.

[25] In step d) of the method of the present invention, in order to form an organic droplet at the end of the inlet portion of the capillary tube (Fig. 2 (b)), it may be possible to apply the reverse pressure necessary to make the droplet, or You can also make drops using vacuum. At this time, there is a space for the volume of the column to exit the column (the capillary column on the opposite side of the ¾ section of the capillary tube) to the column on the outlet side of the capillary. It needs to be filled with a run buffer to maintain electrical contact. In addition, it is necessary to reduce the concentration of salt in the formed organic droplets to a level that allows the subsequent application of LVSEP.

[26] In step e) of the method of the present invention, the sample is extracted from the donor solution (sample solution) into the pentanol droplets for at least 5 minutes (Fig. 2 (b)). To maintain the surface force of the room

It is desirable to apply a predetermined pressure to counteract, and more specifically, the pressure condition can be 0.5 to 1.0 psi for 5 to 20 minutes. By appropriately adjusting the pH of the donor solution (sample solution), It needs to be well extracted into pentanol drops in a weighted state.

[27] In step f) of the method of the present invention, the acceptor solution in the concentrated droplet of sample is removed.

Inject into the capillary and submerge the inlet portion of the capillary into the run buffer. More specifically, the sample extracted by applying hydrodynamically to the acceptor solution in the concentrated droplet is injected into the capillary (Fig. 2 (c)), The hydraulic conditions may preferably be from 2 to 6 psi for 0.5 to 1 minute. At this time, the acceptor solution containing the extracted sample is preferably injected at 5 to 15% of the total capillary volume. Final injection of a sample in excess of 15% of the volume of all capillaries

Not only can the concentration factor be lowered, but also the time required for stacking samples with subsequent application of LVSEP can generally reduce the effectiveness of the method. [28] LVSEP is applied from step g) to the extracted sample injected into the capillary by (SDME) up to step f) of the present invention (FIG. 2 (d)). Acts as an organic memory acceptor for SDME applications and, more specifically, as a sample matrix for LVSEP applications. Applying a reverse voltage to the inlet portion of the capillary when applying LVSEP results in a large electric field on the extraction sample, and at the same time the EOF is directed towards the inlet portion of the capillary, so that the sample matrix is pulled out of the capillary. Stacked in the concentration region between the sample matrix region and the run buffer escaping out of the capillary tube (FIG. 2 (d)).

[29] When applying reverse voltage in step g) of the present invention, the electroosmotic flow (EOF)

Make sure that the EOF is greater than the electrophoretic velocity (EPV) of the sample in the initial LVSEP application, where the sample is heavily injected, but the EOF should be smaller when the separation of the sample matrix occurs. For this purpose, the run buffer of the present invention may be in the form of using a non-aqueous solvent, which preferably lowers EOF significantly, and more preferably, the non-aqueous solvent may be methanol, or for lowering EOF. The run buffer of the present invention may be used by adding an electroosmotic flow modifier (EOF modifier) to a thick or low pH buffer. Therefore, the electrophoretic speed of the concentrated extract sample due to the greatly reduced EOF is By overcoming EOF and advancing towards the detector, they are separated from each other, i.e., LVSEP injects a large amount of sample extracted in a low-conductive solution into the capillary and applies reverse voltage. The matrix of the sample is removed by the EOF toward the sample inlet, whereby a high concentration effect can be expected in such a way that the sample is concentrated while having a high degree of separation.

In step g) of the present invention, while stacking the extracted sample under reverse voltage, the matrix of the sample, pentanol, is removed by the electroosmotic flow toward the inlet portion of the capillary. The run buffer will fill and fill the empty space of the column on the capillary outlet while it is removed to the inlet portion of the capillary by the previous osmosis flow.

[31] In step g) of the method of the present invention, the sample is stacked and capillary electrophoresis is performed.

By observing the current change, the outlet storage container is removed when the change of current occurs suddenly. Observing the current in the capillary electrophoresis during the LVSEP process causes the sample matrix to fall out and the current to change rapidly when the run buffer is filled, removing the run buffer storage container that was placed in the capillary outlet. The rapid increase of the current value is preferably 3.0 μA or more, more preferably 3.0 to 8.0 μA.

In step h) of the present invention (Fig. 2 (e)), the capillary outlet

The run buffer storage container is already removed, and the electrospray ionization

Given the electrospray ionization voltage, the total Because of the magnitude of the voltage, the current value changes slightly after a few minutes. As a result, the concentrated sample, which was stacked in the previous step, moves towards the capillary outlet and is separated by electrophoresis and can be detected by the mass spectrometer. Effects of the Invention

[33] The present invention applies the method of linking both SDME and LVSEP to CE-ESI-MS, significantly improving the concentration factor of the extracted sample compared to individual SDME or LVSEP linkage, and analyzing the sample with high sensitivity. have.

[34] The present invention applies a method of linking both SDME and LVSEP to CE-ESI-MS to provide a low concentration of soil contaminant sample without complex processes.

The concentration factor can be improved significantly and samples can be analyzed with high sensitivity.

Brief description of the drawings

1 is a schematic diagram of the device features applied to the method of the present invention.

2 is a schematic diagram for explaining a schematic description of the method of the present invention.

In FIG. 3, (a) shows a mass electropherogram following application of conventional CE-ESI-MS for 5 μΜ samples, and (b) SDME-LVSEP of the present invention for 10 nM samples. -Mass electropherogram is shown following the application of CE-ESI-MS. Sample peak: (1) MCPA; (2) 2,4,5-trifluorobenzoic acid; (3)

2,3,4,5-tetrafluorobenzoic acid; (4) 4,6-DNOC; (5) 2,4-DNP

4 shows the present invention for an actual soil sample containing a pollutant sample.

Mass electropherogram is shown following the application of SDME-LVSEP-CE-ESI-MS. Just before applying SDME-LVSEP-CE-ESI-MS, each of the samples

The spiked concentration of the analyte is 20 g / L. Sample peaks: (1) MCPA; (2) 2, ^4, 5-trifluoro-benzoic acid; (3) 2,3,4,5-tetrafluorobenzoic acid; (4) 4,6-DNOC; (5) 2,4-DNP

Mode for Carrying Out the Invention

[39] Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples. Changes to substitutions and other equivalent embodiments without departing from the technical spirit will be apparent to those of ordinary skill in the art.

[40]

[41] <Sample Preparation>

[42] 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, and

2,4-dinitrophenol was purchased from Aldrich (Milwaukee, WI, USA).

(4-Chloro-2-methylphenoxy) acetic acid (MCPA) was obtained from TCI (Tokyo, Japan).

Purchased. 2-methyl-4,6-dinitrophenol (4,6-DNOC), ammonium formate, sodium hydroxide, ammonium hydroxide solution (ammonium hydroxide solution, ammonium acetate, hydrochloric acid,

HPLC-gmde methanol, HPLC grade

Pentanol (1-pentanol) (HPLC-grade 1-pentanol), and HPLC grade

Isopropyl alcohol (HPLC-grade isopropyl alcohol) is Sigma (St. Louis, Mo,

USA). Deionized water was Milli-Q.

Prepared using the system (Millipore, Bedford, Mass., USA).

[43]

[44] <CE-ESI-MS>

[45] The basic form of the capillary has a total length of 100 cm, 50 μηι ID, and 280 μι OD

Uncoated fused silica capillary (Postnova, Landsberg am Lech, Germany) and the capillary was washed with Ol M NaOH, water and run buffer at 80 psi for 10 minutes each. The run buffer was 25 mM ammonium formate adjusted to pH 8.0 with methanol. (ammonium formate). Capillary cartridges were set at 25 ° C.

[46] CE was performed using Beckman's MDQ CE system with 32 Karat software (Fullerton, CA, USA), and electrophoresis was performed with a reverse voltage of -22 kV. MS uses triple quadrapole mass spectrometers (Quattro LC, Waters-Micromass,

Manchester, UK) with Masslynx 3.3 software

Adjusted. In MS, a sheath liquid of 2 mM ammonium acetate in an isopropyl alcohol / water (80:20 v / v) mixed solvent was held by a syringe pump at a rate of 1.0 μΙ 7ιηίη. Cone voltage of 20 V was used in negative ionization mode in MS. The ionization source block temperature in MS was set to 80 ° C, and nitrogen gas at 20 IJh flow rate was used as the nebulizer gas. The ESI voltage was set to -2.5 kV.

[47] The analyte was a mixture of 0, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP. The solution was injected into the capillary at 1 psi pressure for 10 seconds while dissolved in a buffer of 5 μΜ.

MS spectrum data for CE-ESI-MS of analytical samples were recorded under multiple reaction monitoring (MRM) mode in the range of m / z 20-500 (Table 1), and the mass electropherogram was observed (Fig. 1). 3 (a)). [Table 1]

[52] <SDME-LVSEP-CE-ESI-MS: Example 1>

[53] The capillaries Length ^{100 cm, 50 μ η ι ID} , and 280 μ ^η ι said fused silica capillary (Postnova, Landsberg am Lech, Germany) 0] with the OD, the run buffer used to be used has a methanol pH 25 mM ammonium formate (ammonium)

formate).

[54] Prior to full-scale application of SDME, the capillary was washed with 1 M NaOH and water for 10 minutes, and the inlet portion of the capillary was hydrophobic support Teflon sleeve (500 [xm OD, Zeus, SC, US A)). (Fig. 1).

[55] In the full-scale application of SDME, the inlet portion of the capillary is coated with Teflon sleeve.

The run buffer was filled and filled. Also, the capillary outlet (¾) on the orifice side of the mass spectrometer (MS) was placed in an outlet reservoir containing the run buffer to lock the capillary outlet.

Next, the organic acceptor pentanol was injected into the capillary at 10% (180 nL) of the total capillary volume (Fig. 2 (a)), and the inlet portion of the capillary was removed from the donor solution (0.1 M HC1 acidic aqueous solution). : The donor solution (sample solution) contains the analytical sample, and the analytical sample is soil contaminant, MCPA, 2,4,5—trifluorobenzoic acid, 2,3,4,5-tetra Consisting of fluorobenzoic acid, 4,6-DNOC and 2,4-DNP

It was a mixture (concentration in donor solution: 10 nM). At pH 1.0, which is the pH condition of the donor solution, it was confirmed that the contaminants were mainly in a neutral state.

[57] Next, a reverse pressure was applied at 2 psi for 20 minutes to draw the pentanol injected into the capillary toward the inlet portion of the capillary to form an organic drop at the end of the capillary inlet portion. The empty space was filled with the run buffer of the outlet storage container. The analysis sample was extracted from the donor solution (sample solution) for 10 minutes with the pentanol organic droplet formed at the end of the inlet portion of the capillary tube (Fig. 2 (b)). 10 minutes at 0.8 psi

By counteracting the surface force, the stability of the room in the extraction process was maintained.

[58] Next, the sample was concentrated in an acceptor solution in a concentrated pentanol drop for 5 minutes. The analytical sample extracted by applying hydrodynamically to psi was injected into the capillary (FIG. 2 (c)), and the acceptor solution was injected into the capillary at 10% of the total capillary volume. Specifically, the storage vessel, which is filled with the capillary portion of the capillary, changes from the donor solution reservoir to the run buffer storage vessel.

[59] Next, an analysis was performed by applying a reverse voltage of -22 kV to the inlet portion of the capillary.

The application of LVSEP was started by stacking the samples (Fig. 2 (d)), and the pentanol of the acceptor solution injected into the capillary acted as a sample matrix for LVSEP application. With the application of LVSEP, the EOF is directed towards the inlet portion of the capillary, so the sample matrix, pentanol, is pulled out of the capillary, which also fills the empty space of the column on the capillary outlet with the run buffer of the outlet reservoir. The extracted analyte sample is stacked in the concentration region between the sample matrix area and the run buffer escaping out of the capillary (Fig. 2 (d)), and the current of capillary electrophoresis is 5.5 土 0.3 μΑ at about 0 μA in 13 ± 2 minutes. The sample stacking was completed by manually removing the outlet storage container and rapidly confirming that the current of the capillary electrophoresis changed rapidly. Sample stacking was based on the time when the acceptor solution was injected into the capillary. It was completed in minutes.

[60] Next, interworking with CE-ESI-MS was established. CE 32 Karat Software

The electrophoresis was performed using Beckman's MDQ CE system (Fullerton, CA, USA), and electrophoretic separation of the samples was carried out using a voltage of -22 kV under 0.7 psi pressure. MS performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK) with Masslynx 3.3 software.

Adjusted. In MS, a sheath liquid of 2 mM ammonium acetate in an isopropyl alcohol / water (80:20 v / v) mixed solvent was flowed at a rate of 1.0 IJ min by a syringe pump. Cone voltage of 20 V was used in negative ionization mode in MS. The ionization source block temperature in MS was set to 80 ° C, and nitrogen gas at 20 IJh flow rate was used as the nebulizer gas. The ESI voltage was set to -2.5 kV. With the application of EST voltage, the stacked concentrated sample is MCPA,

2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and

In the mixture consisting of 2,4-DNP, each individual substance is dissociated into anion ions from which hydrogen ions have been separated. Thus, a composite sample of each of the individual substances dissociated with anion ions is capillary outlet ^ It was separated by electrophoresis while moving toward), and was detected by a mass spectrometer (FIG. 2 (e)), and accordingly, the mass electropherogram was observed (FIG.

[61] Mass for SDME-LVSEP-CE-ESI-MS of the present invention according to FIG. 3 (b).

In the electropherogram (Figure 3 (b)), the peak shift for each analyte The migration time is observed to be 15 minutes longer than that of CE-ESI-MS (Fig. 3 (a)). In the present invention, 15 minutes is required for stacking samples according to the application of LVSEP. It is said that this causes the temporal difference.

[62]

[63] <Comparison of enrichment factor (EF) of samples according to SDME-LVSEP-CE-ESI-MS, LVSEP-CE-ESI-MS and SDME-CE-ESI-MS>

[64] Examination Example 1 (Current SDME-LVSEP-CE-ESI-MS): As described above, the present invention applies the SDME-LVSEP-CE-ESI-MS of the present invention.

A mixture of 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC, and 2,4-DNP. The analytical sample is a donor solution with a concentration of ΙΟ ηΜ. Dissolved in.

Comparative Example 1 (예 LVSEP-CE-ESI-MS): Not applicable to SDME and analysis of samples

Except for the different concentrations, basically the same method as in Example 1 was applied. The analytical sample was the same material as in Example 1. The run buffer was also set to pH 8.0 with methanol as in Example 1. 25 mM ammonium formate. The analytical sample was dissolved in pentanol at a concentration of 200 nM, whereby the pentanol solution containing the analytical sample was injected into the capillary at 10% of the total capillary volume. The sample was stacked by applying a reverse voltage of -22 kV, and after stacking, the same CE-ESI-MS operating conditions as in Example 1 were applied.

Comparative Example 2 rSDME-CE-ESr-MS): No application of LVSEP and analysis of samples

Except for the different concentrations, basically the same method was applied as in Example 1. The analytical sample was the same material as in Example 1. The run buffer was also set to pH 8.0 with methanol the same as in Example 1. 25 mM ammonium formate. The analytical sample was dissolved in a donor solution (0.1 M HC1 acid aqueous solution) at a concentration of 100 nM. As the organic acceptor for extracting the analytical sample, the same pentanol as in Example 1 was used. Next, in the same manner as in Example 1, an analytical sample was extracted from the donor solution for 10 minutes with a pentanol organic drop formed at the end of the inlet portion of the capillary, and the CE-EST-MS operation after the extracted sample was injected into the capillary tube. The conditions were the same as in Example 1.

In contrast with the conventional CE-ESI-MS application, the concentration factors of the samples according to the application of the respective method of Example 1 and the comparative examples are shown in Table 2 (compare the concentration factors = 4). It is written in.

[68] [Table 2]

Once described in Table 2, a concentration coefficient of several hundred to thousand thousand folds is found in the application of the method of Example 1 of the present invention. The concentration coefficient of scores-fold is only confirmed. Thus, as in the contrast of Table 2, the concentration coefficient values for each individual substance constituting the analytical sample in Example 1 of the present invention are Compared with the comparative examples, it is confirmed that the improvement is more than 10 times. At this time, considering that the concentration of the analytical sample in Example 1 of the present invention is a low concentration of 10 nM, the improved coefficient as described above shows the phenomena of the present invention. This is further supported by the synergistic effect of double concentrations in the application of the method of the present invention, in which both SDME and LVSEP are interlocked, especially if the analytical sample is a contaminant that can be contained in the soil. Low concentration soil pollution by the method of the invention Along the sample to be highly concentrated factor can be analyzed with high sensitivity.

For the actual soil samples containing contaminant samples

Application of SDME-LVSEP-CE-ESI-MS: Example 2>

Analytical samples included in 0.5 g soil sample were MCPA, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP. It is a complex made up. The soil sample containing the analytical sample was spiked with a standard solution and then completely dried at room temperature overnight. Next, 10 mL of water was added to the soil sample and the resulting mixture was added for 30 minutes.

Ultrasonicated 1 mL of the upper solution was diluted to 0.2 mL HC1 and adjusted to 2 mL.

Soiled spiked soil samples (concentration level: 50 nM)

SDME-LVSEP-C & ESI-MS was applied (just before the application of SDME-LVSEP-CE-ESI-MS, spiked concentration of each analyte included in the sample

concentration) is 20 g / L). In the application of SDME-LVSEP-CE-ESI-MS, LVSEP According to the sample stacking time was 20 minutes, accordingly the peak migration time for each analyte in the mass electropherogram (Fig. 4)

It was observed that the CE-ESI-MS was about 20 minutes longer than that shown in FIG. 3 (a). In Example 2, the same method as in Example 1 was applied except for the donor solution and the sample stacking time, and the mass electropherogram (Fig.

As confirmed by 4), high-sensitivity analysis was possible for low concentration analytical samples included in real soil samples.

[75]

[76]

Claims

Claim

[Claim 1] a) Filling the run buffer into the capillary;

b) placing an outlet storage container with a run buffer in the capillary outlet on the orifice side of the mass spectrometer (MS) to lock the capillary outlet;

c) injecting the organic acceptor pentanol into the capillary and dipping the inlet portion of the capillary into the donor solution (sample solution);

d) drawing pentanol injected into the capillary toward the inlet portion of the capillary to form an organic chamber at the end of the inlet portion of the capillary; e) extracting the sample from the donor solution into the droplet; f) injecting the acceptor solution in the concentrated droplet of the sample into the capillary and dipping the inlet portion of the capillary into the run buffer;

g) stacking the extracted sample by applying a reverse voltage to the inlet portion of the capillary and observing the current change in capillary electrophoresis to remove the outstorage reservoir when the current change rapidly occurs; and

h) removing the sheath liquid from the mass spectrometer and applying an electrospray ionization voltage (ESI voltage) to move the stacked concentrate sample to the mass spectrometer; Single drop microscopy (SDME) and large volume piece stacking method (LVSEP) using an electroosmotic flow pump, characterized by including

CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometry)

By interlocking, continuous stacking, separation and analysis of samples extracted from organic matter.

[Claim 2] In Section I,

Characterized in that the inlet portion of the capillary is coated with a Teflon sleeve, a hydrophobic support.

[Claim 3] In paragraph 1,

In step c), the sample applied to the donor solution is characterized in that the soil contaminants.

[Claim 4] In paragraph 3,

PH of said donor solution is 0.5-2.

[Claim 5] In paragraph 1,

Characterized in that the run buffer supplements the empty space of the column toward the capillary outlet during the formation of the droplet in step d).

[Claim 6] In paragraph 1,

And in step g), characterized in that the electroosmotic flow (EOF) is less than the electrophoretic velocity of the sample.

[Claim 7] In paragraph 6,

The run buffer is characterized by using a non-aqueous solvent as a buffer.

[Claim 8] In paragraph 7,

The non-aqueous solvent is characterized in that the methanol.

[Claim 9] In paragraph 6,

Wherein said run buffer is characterized by the use of an electro-osmotic flow modifier (EOF modifier) in a neutral or low pH buffer.

[Claim 10] In paragraph 1,

In step g), while stacking the sample by applying a reverse voltage, the matrix of the sample, pentanol, is discharged from the capillary by electroosmotic flow.

Characterized by being removed towards the inlet portion.

[Claim 11] In paragraph 10,

Characterized in that the empty space buffer of the column at the capillary outlet is complemented while the pentanol is removed towards the inlet portion of the capillary by an electroosmotic flow.