WO2009011488A1

WO2009011488A1 - Large-volume stacking method using the electroosmotic flow pump in capillary electrophoresis interlocked with mass spectrometry and the equipment for it

Info

Publication number: WO2009011488A1
Application number: PCT/KR2008/002269
Authority: WO
Inventors: Du Soo Chung; Ji Hye Kim
Original assignee: Seoul National University Industry Foundation
Priority date: 2007-07-14
Filing date: 2008-04-22
Publication date: 2009-01-22
Also published as: KR101144974B1; KR20090007519A

Abstract

This invention involves an apparatus of outlet reservoir and a technique for performing large volume stacking using electroosmotic flow pump (LVSEP) in capillary electrophoresis-mass spectrometry (CE-MS), which is a simple method to achieve greater enhancement in detection sensitivity. This invention utilizes an apparatus which is placed between MS orifice and capillary outlet and allows to supply the flow of the run buffer in the direction opposite to sample injection during sample stacking. The capillary outlet and electrospray ionization emitter are immersed into the apparatus which is filled with the run buffer. This apparatus is in a manner that attachment and detachment are allowed between MS orifice and capillary outlet.

Description

[DESCRIPTION] [Invention Title]

LARGE-VOLUME STACKING METHOD USING THE ELECTROOSMOTIC FLOW PUMP IN CAPILLARY ELECTROPHORESIS INTERLOCKED WITH MASS SPECTROMETRY AND THE EQUIPMENT FOR IT [Technical Field]

This invention relates to apparatus for accomplishing separation, enhancing of detection sensitivity, stacking in capillary electrophoresis - mass spectrometry, and more particularly to large-volume stacking method using the electroosmotic flow pump (LVSEP) in capillary electrophoresis combined with mass spectrometry. [Background Art]

Recently there has been an increased interest in coupling capillary electrophoresis (CE) with mass spectrometry (MS) to take advantage of the high performance separation of CE and the rapid identification and structural characterization of MS. However, the concentration sensitivity of CE/MS is often less than needed due to the nanoliter injection volumes. In particular, the sheath liquid interface that is the most widely applied in CE/MS can degrade the sensitivity because analyte is diluted by a sheath liquid. In the proteomic and metabolomic analysis, it is required to increase the detection sensitivity for the quantification.

Large-volume sample stacking using the electroosmotic flow pump (LVSEP) is one of the most convenient and useful on-line stacking techniques used in CE. A reverse voltage is applied across a capillary filled with a low- conductivity sample. Then anionic analytes, for example, are stacked at the boundary between the sample zone and the high-conductivity run buffer zone which is moving backwards to the inlet side by the electroosmotic flow (EOF). If the magnitude of EOF for a capillary filled with the run buffer is smaller the electrophoretic speeds of the anionic analytes, they will overcome the EOF and migrate forward to the detector before removed from the capillary. Thus the sample matrix plug causing adverse effects on the separation is pumped out of the capillary without loosing analytes, commonly providing sensitivity enhancement factors of several hundred-fold. To accomplish LVSEP, EOF must be smaller than the electrophoretic velocity. For stacking anions, an EOF modifier may be added to a run buffer or low pH buffer may be used. For stacking cations, EOF is suppressed and the direction of EOF should also be reversed with a low pH buffer containing a low concentration of cationic surfactant or using specially coated capillaries.

Although LVSEP is a promising technique, its application to CE/MS has not been easy due to the lack of an outlet reservoir in conventional CE/MS interfaces to supply the reverse flow of run buffer for pumping out the sample matrix. Recently, Chen et al . in Analytical Chemistry,75 (2003) pages 503-508, reported large volume stacking in CE/MS using a low-flow sheath liquid CE/ESI-MS interface which can be provide a reverse flow of buffer from a microcentrifuge reservoir integrated in their interface. In order to make employing LVSEP to CE/MS even easier, inventor presents a quite simple method of supplying the reverse flow of run buffer. By placing a small centrifuge tube filled with a run buffer during the stacking process at the outlet end of a CE/MS interface, the reverse flow of run buffer needed for the EOF pump is supplied.

[Disclosure]

[Technical Problem]

The present invention is a simple method for LVSEP CE/MS and apparatus for supplying the solution from the outlet reservoir. A buffer vial filled with a run buffer is placed at the capillary outlet during stacking. After stacking, the buffer vial is removed for CE/MS analysis. The use of an intact commercial sheath liquid interface provides a stable spray and makes this method to be easily applicable. The sensitivity enhanced more than 2 orders of magnitude.

Therefore, to perform even easier the coupling of LVSEP and CE/MS, one is that the interface of MS is needed some modification for supplying the run buffer. Another is a usage of reduced the EOF. The inventor has reported LVSEP using methanol as a run buffer solvent with bare fused silica capillary in Electrophoresis, 23 (2002) page 49-55. It can be possible to reduce the EOF simply and also to facilitate the analysis of hydrophobic compounds due to higher solubility in organic solvents. Furthermore, the property of methanol is volatile can produce the stable electrospray in CE/MS._. [Technical Solution]

This invention provide an analytic method in employing large volume stacking using an electroosraotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS), wherein the method comprises the step of supplying the run buffer by placing a small outlet reservoir filled with a run buffer at the outlet end of a CE/MS interface during the stacking process. Also this invention provide an analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS)), which comprises the steps of: injecting the lower conductivity sample solution than a run buffer into a capillary; placing a outlet reservoir filled with a run buffer in the capillary; immersing the capillary outlet into run buffer by placing outlet reservoir filled with a run buffer at capillary outlet; applying a reverse voltage for a stacking and separation; removing the outlet reservoir when a electric current is abruptly increased in CE; delivering a sheath liquid and applying an ESI voltage; detecting the stacked sample in mass spectrometer.

Also, this invention is an analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS) according to above, wherein the electroosmotic flow(EOF) in the step of applying the reverse voltage is smaller than the electrophoretic velocity of the sample solution, and wherein if the sample is anionic, the buffer is nonaquous solvent or EOF modifier is added to low pH buffer, and if the sample is cationic, cationic surfactant is added to the buffer or the capillary is coated, and wherein the buffer is organic solvent such as methanol.

This invention can provide capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus, wherein the interface apparatus has a configuration with an outlet reservoir which can be attached and detached between MS orifice and capillary outlet, and the capillary outlet immerses into the run buffer of outlet reservoir. This invention is capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus according to above, wherein said outlet reservoir has a tiny hole which allows the capillary outlet to insert into the outlet reservoir, and wherein said outlet reservoir has a tiny hole which allows the capillary outlet to insert into the outlet reservoir.

This invention is outlet reservoir used at capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus, wherein the outlet reservoir includes a hole for inserting a capillary outlet wherein a position of the hole is the side the outlet reservoir, and the outlet reservoir according to above, wherein the hole of the outlet reservoir is sealed by a rubber seal wherein the center area of the rubber seal includes a perforated hole that allows the capillary outlet to insert into the outlet reservoir and wherein said the rubber seal includes different diameter, one is middle part that has a smaller diameter and the other diameters have larger diameter than middle at oneside or both side of middle part.

[Advantageous Effects]

This invention provides a simple method to perform LVSEP in CE/MS using a outlet reservoir during sample stacking. Although LVSEP is a promising technique as a stacking method, its application to CE/MS has not been easy due to the lack of^' an outlet reservoir in conventional CE/MS interfaces to supply the reverse flow (toward the inlet vial) of run buffer for pumping out the sample matrix. In order to make employing LVSEP to CE/MS easier, this invention presents a quite simple method of supplying the reverse flow of run buffer. By placing a small centrifuge tube (as a outlet vial) filled with a run buffer during the stacking process at the outlet end of a CE/MS interface, the reverse flow of run buffer needed for the EOF pump is supplied. The sensitivity enhancement factors are several hundred-fold for the anionic analytes as usually obtained with LVSEP. This simple' scheme does not disturb the electrospray geometry and does not require liquid exchange procedure needed for the elaborate low-flow sheath liquid CE/ESI-MS interface. Since this disposable outlet reservoir can be made without any special tool or skills and does not modify the existing interface at all, this scheme can be easily applied to a CE/MS instrument shared as a common facility.

Furthermore, when the CZE/MS as a conventional method is used to detect some samples, detection is not accomplished with some concentration of samples. However, when this invention method is used to detect the same concentration of samples, by achieving the sensitivity improvement, samples is detected in LVSEP CE/MS. This invention is capable of the detection of low concentration (e.g. low concentration of metabolites, environment samples) in CE/MS with a high efficiency of CE and a structural elucidation of MS.

The coupling LVSEP to CE/MS is even easier to be employed the preferable outlet reservoir. [Description of Drawings]

FIG. 1 is a schematic illustration of LVSEP CE/MS system or CE/MS system with the present invention. FIG. 2 is an electric current profile of LVSEP CE/ESI-MS; (1) stacking and pumping the sample matrix plug out of the capillary, (2) completion of LVSEP, and (3) ESI voltage application for CE/MS.

FIG. 3 is MS electropherograms for six anion analytes in LVSEP CE/MS with the present invention.

FIG. 4 is a schematic illustration of LVSEP.

FIG. 5 illustrates an embodiment of the present invention.

FIG. 6 is an illustration of capillary electrophoresis-mass spectrometry interface made in accordance with the invention.

FIG. 7 is an illustration to make an outlet reservoir.

FIG. 8 illustrates an embodiment of the rubber sealing in outlet reservoir. [Best Mode]

The present invention describes in detail herein. To provide a better understanding this invention, axiomatic system such as ESI-MS, ESI-MS interface, and sheath liquid interface is omitted.

FIG. 4 illustrates the theory of LVSEP of anions. (1) filling the whole capillary with the sample, (2) removal of sample matrix plug during sample stacking under a reverse potential, and (3) separation of the highly stacked sample. LVSEP is done by injecting large-volume sample solution, which is dissolved in a low conductivity solution (e.g. water or extremely diluted buffer), into a capillary hydrodynamically and applying reverse polarity across the capillary. Because the low concentration sample solution has a higher resistivity, the electric field strength in the sample solution is higher than that in a run buffer. Consequently, the sample ions (anions) migrate more rapidly in the sample solution than those migrate in a run buffer, and anions experience slow down at the boundary between^' sample plug and a run buffer and then they are stacked at the front of the sample solution. While anions migrate toward the outlet due to their mobility, the direction of EOF is toward the inlet. To use the EOF as a pump, the EOF should be low enough to allow the analytes to migrate toward the outlet. Suppressed the EOF can be easily achieved by using nonaqueous solvent such as methanol. Sample matrix is removed from the capillary with reduced the EOF using methanol buffer, at the same time analytes are stacked, and then they are separated and migrate to the detector. Because sample matrix is pumped out from the capillary, LVSEP can preserve high resolution. Hundred-fold concentration factor are reported for LVSEP.

Large-volume sample stacking using the electroosmotic flow pump (LVSEP) is one of the on-line stacking techniques used in CE for enhancing sensitivity. As followed the LVSEP theory, EOF must be smaller than the electrophoretic velocity. For stacking anions, an EOF modifier may be added to a run buffer or low pH buffer may be used. For stacking cations, EOF is suppressed and the direction of EOF should also be reversed with a low pH buffer containing a low concentration of cationic surfactant or using specially coated capillaries. The substances about the reducing EOF are omitted since a lot of research have been reported. The inventor has reported LVSEP using methanol as a run buffer solvent with bare fused silica capillary. It can be possible to reduce the EOF simply and also to facilitate the analysis of hydrophobic compounds due to higher solubility in organic solvents. Furthermore, the property of methanol is volatile can produce the stable electrospray in CE/MS.

LVSEP hasn' t connected to CE/MS well. Since a outlet reservoir was not usually used in CE/MS, It cannot supply the flow of support buffer in the direction opposite to sample injection during the sample stacking process. A small vial (such as a PCR vial) for the outlet reservoir is to place between MS orifice and capillary outlet and filled with the run buffer. It is placed to put capillary outlet into the outlet reservoir

FIG. 5 illustrates the coupling of outlet reservoir with an ESI sprayer. FIG. 5 as an example, show how to insert the capillary and ESI emitter into the run buffer of outlet reservoir. A rubber seal has a perforated center which can be inserted with a capillary and ESI emitter. ESI emitter is made of stainless steel. A capillary outlet is surrounded by a stainless steel tube. This stainless steel tube as a tip is a electrode herein. Therefore a capillary outlet and tip should be contacted with the run buffer of outlet reservoir to achieve the electric contact. Generally the outlet of a CE capillary extend beyond the sheath liquid tube (herein, ESI emitter or tip) by 0.5 ~ 2 mm. The inserted length of capillary and tip can be different depending on the outlet reservoir size, rubber seal size, and the length of ESI emitter. The configuration method between outlet reservoir and MS interface can be tolerant to the variation in MS interface conditions.

For the preferable fixing the outlet reservoir at MS interface, a bar is attached to the outlet reservoir. In outlet reservoir, a cap is needed to prevent inside the solution form evaporating. The run buffer inside the reservoir does not leak and most of the volume inside can be used. This outlet reservoir is useful on horizontally alined ESI, especially. This invention also can be applied to many CE separation mode.

FIG. 6 is a picture which is employed with a FIG. 5 provides a better understanding of the present invention. FIG. 7 is a fabrication process of outlet reservoir. A flexible rubber seal of a dumbbell shape is trimmed from an MDQ capillary cartridge (#144866, Beckman, Palo Alto, CA, USA). A rubber sealing is tolerant to the variation in outlet reservoir. That means, to achieve the sealing, the size and the brand is not critical. FIG. 8. is a detailed illustration of a trimmed rubber seal. The trimmed rubber seal consists of two kinds of diameter. Both ends of a rubber seal have a lager diameter than the middle part of it. At sides of view, a rubber seal is perforated for inserting a capillary and ESI emitter. To achieve the sealing, a size and the brand is not critical, however, illustrations of FIG. 7 and 8 can be used as a preferable form.

To achieve the electric contact, capillary outlet and ESI emitter are immersed into the run buffer of outlet reservoir through the rubber sealing. FIG. 8. is illustrated the embodiment of the rubber seal which is used to seal a hole of the outlet reservoir and of which center part is consisted of the tiny hole to insert a capillary.

The process of LVSEP can be verified by monitoring the electric current. The electric current is almost zero at the initial run, which increases abruptly to 8.5 μA near 15 min. (This experimental conditions provide a better understanding of the present invention. The values can be changed depending on the experimental conditions). This means the stacking process has been finished. At this time, the outlet reservoir is removed from the capillary outlet. Then the electric current is decreased from 8.5 μA to around 7.5 μA due to decrease of total electric voltage across the capillary. As shown the current profile of CZE/MS and LVSEP/MS in FIG 2, the electric current is constant to 7.5 μA in CZE/MS, which becomes the same electric current at the moment that analytes are separated after preconcentration in LVESP/MS. During LVSEP process, setting the run buffer reservoir at the capillary outlet is more easer than using the low-flow interface as reported. Instead of making a new device for LVSEP/MS, it has only to use a run buffer reservoir at the capillary outlet, and this method can be applied to most mass spectrometer. This method does not affect an ESI stability because a spray tip position is not almost changed, and stacking and separation processes can occur successively.

[Mode for Invention]

The features of the invention described in detail below.

The fabrication process of the outlet reservoir and its coupling to MS are as follows.

An one side of small vial (for example, 200- μL snap cap tube (Molecular BioProducts, CA, U.S.A.)) is drilled 3 mm hole at a 0.5 cm away from the bottom of tube using a drill. Then, the rubber seal (for example, #144866, Beckman, Palo Alto, CA, U.S.A.) is prepared by cutting. Dumbbell shaped rubber seal has two different diameter sizes. Both ends of the rubber seal diameters are approximately 4 mm. Middle part of the seal is approximately 3 mm. A cutting length of the rubber seal is approximately 2.5 mm including the middle part. (In this case, The length of 2.5 mm can be changed depending on the ESI emitter) Final step, the trimmed rubber seal is popped in and a approximately 10 cm bar is attached to the tube as a grip. Herein is used a 10 cm-length bar, but the length of a bar can be adjusted preferably. There is a perforated hole at the center of the rubber seal which is used to insert a capillary. The capillary outlet and electrospray ionization emitter are immersed into the apparatus which is filled with the run buffer. Then snap cap or cap of outlet vial is closed to prevent inside the solution from evaporating. That is the reason to use the cap in this invention.

Chemicals and instruments are used as follows to show the invention as an example.

Ammonium formate is obtained from Sigma(St. Louis, MO, USA), Tetrachlorophenol and Pentachlorophenol were obtained from Aldrich (Milwaukee, WI, USA). Sodium hydroxide, and ammonium acetate were from Sigma (St. Louis, MO, USA). Methanol and isopropyl alcohol of HPLC grade were from Mallinckrodt Baker (Paris, KY, USA). Deionized water was prepared with a MiHi-Q system (Millipore, Bedford, MA, USA).

CE was performed using a CE system P/ACE MDQ and 32 Karat Software. The capillaries (Polymicro Technologies, Phoenix, AZ, USA) were uncoated fused- silica capillaries with a total length of 90 cm and 50 m ID. UV detection is not used in connecting MS.

Separation voltage is -20kv, and the capillary is filled with sample adn sample is dil luted with methanol. The run buffer was ammonium formate in methanol. The apparent pH of the buffer was adjusted to 8.0. Mass spectrometry was Q-TOF Ultima Global(Waters-Micromass, Manchester, UK) equipped with a Z-Spray interface, performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK) equipped with a Z-Spray interface in the negative ionization mode. And sheath liquid is 80% isopropyl alcohol at at a flow rate of 1.2 ul/min. The ionization source block temperature was set to 8OC, and desolvation gas flow is at 50L/hr, nitrogen gas at about 20psi was used as a nebulizer gas. electropray voltage is -2.4 - 2.8kv.

After capillary was filled with the run buffer and reversed voltage of -20 kV was applied with lpsi at the same time sheath liquid was introduced, and electrospray voltage was applied. On monitoring the total ion current, the capillary for the ESI spray was positioned where the stable spray was continued. After the spray positioning, sheath flow was stopped, electrospray voltage and CE separation voltage were applied to 0 kV. A large volume of sample, dissolved in methanol, was injected hydrodynamically into the capillary. Then the inlet vial filled with 20 mM ammonium formate buffer pH 8.0. 200-ml vial for the outlet reservoir was to place between MS orifice and capillary outlet and filled with the run buffer. 9-cm bar as a grip was attached to the outlet reservoir. The shape of the outlet reservoir was similar to scoop. It was placed to put capillary outlet into the outlet reservoir in FIG 1. Using the bar as a grip, scoop-shaped outlet reservoir was fixed to the frame with adhesive tape. Then CE separation voltage was applied to -20 kV. At the same time, the sheath liquid was not supplied and electrospray voltage was applied to 0 kV. Therefore the total ion current (TIC) was zero because there was no ion to enter the MS. After high voltage was applied across the capillary, anionic analytes were stacked at the front of the sample plug. The direction of EOF was reversed for the direction of migrating analytes. Sample matrix was removed by EOF pump and the run buffer was supplied from the outlet into the capillary column. Sample stacking process can be monitored with CE current. The electric current was almost 0 in initial state of the LVSEP. After 20 min, electric current increased abruptly and reached to 7 mA as the same electric current of CZE. At this time, the stacking process has been finished, and it needs to remove the run buffer vial from the outlet. The capillary is filled with the run buffer, samples are stacked. Thereafter LVSEP/MS process has only to perform the conventional CZE/MS. Additionally sheath liquid is supplied and nebulizer gas is supplied. Electrosoray voltage is applied and the spectrum is detected in the negative ion mode. Then samples are separated and detected according to their mobility. TIC (total ion current) is not recorded in MS chromatogram until LVSEP/MS is performed such as the CZE/MS, after sample stacking, n figure 3, 50nm sample in methanol is used, and separation voltage is applied to -20 kV at 20 mM ammoniumformate pH 8.0, and that show good sensitivity of clolophenol in mass electoropherogram.

This LVSEP/MS system is simple stacking method in MS detection. It needs to remove the outlet run buffer vial, after sample stacking, which is placed from the capillary outlet before run. Then the remaining of LVSEP/MS procedure is only performed such as sheath flow CZE/MS. In addition, this method is not concerned about reducing the ESI stability because the outlet vial is only moved without changing the original position of the outlet capillary. Large volume stackingusing an electroosmotic flow pump (LVSEP) is one of the most convenient and useful on-line stacking techniques for CE. LVSEP has been applied to detect the drug, metabolites, pesticide and other compounds. LVSEP has been usually coupled to UV detection system. However, LVSEP can be combined with MS system (e.g. Tandem MS, time of flight MS) even easier, using the present invention.

The features of the invention is not limited to what is described in above examples but also includes undescribed equivalents of the invention that is recited in the claims.

Claims

[CLAIMS] [Claim 1]

An analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS), wherein the method comprises the step of supplying the run buffer by placing a small outlet reservoir filled with a run buffer at the outlet end of a CE/MS interface during the stacking process.

[Claim 2]

An analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS)), which comprises the steps of: injecting the lower conductivity sample solution than a run buffer into a capillary; placing a outlet reservoir filled with a run buffer in the capillary; immersing the capillary outlet into run buffer by placing outlet reservoir filled with a run buffer at capillary outlet; applying a reverse voltage for a stacking and separation," removing the outlet reservoir when a electric current is abruptly increased in CE; delivering a sheath liquid and applying an ESI voltage; detecting the stacked sample in mass spectrometer. [Claim 3]

An Analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS) according to claim 2, wherein the electroosmotic flow(EOF) in the step of applying the reverse voltage is smaller than the electrophoretic velocity of the sample solution. [Claim 4]

An Analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS) according to claim 3, wherein if the sample is anionic, the buffer is nonaquous solvent or EOF modifier is added to low pH buffer, and if the sample is cationic, cationic surfactant is added to the buffer or the capillary is coated. [Claim 5]

An Analytic method in employing large volume stacking using an electroosmotic flow pump (LVSEP) to capillary electrophoresis/mass spectrometry (CE/MS) according to claim 3, wherein the buffer is organic solvent such as methanol. [Claim 6]

Capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus, wherein the interface apparatus has a configuration with an outlet reservoir which can be attached and detached between MS orifice and capillary outlet, and the capillary outlet immerses into the run buffer of outlet reservoir. [Claim 7]

Capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus according to claim 6, wherein said outlet reservoir has a tiny hole which allows the capillary outlet to insert into the outlet reservoir. [Claim 8]

Capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus according to claim 6, wherein said outlet reservoir has a tiny hole which allows the capillary outlet to insert into the outlet reservoir. [Claim 9]

Outlet reservoir used at capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus, wherein the outlet reservoir includes a hole for inserting a capillary outlet wherein a position of the hole is the side the outlet reservoir. [Claim 10]

Outlet reservoir used at capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus according to claim 9, wherein the hole of the outlet reservoir is sealed by a rubber seal wherein the center area of the rubber seal includes a perforated hole that allows the capillary outlet to insert into the outlet reservoir. [Claim 11]

Outlet reservoir used at capillary electrophoresis/mass spectrometry (CE/MS) interface apparatus according to claim 10, wherein said the rubber seal includes different diameter, one is middle part that has a smaller diameter and the other diameters have larger diameter than middle at oneside or both side of middle part.