JP4613002B2 - Method for manufacturing column-integrated chip for electrospray - Google Patents

Description

  The present invention relates to a liquid chromatograph / mass spectrometer (LC / MS), and more particularly, to an LC / MS that analyzes a solution containing a sample such as a peptide or protein at a micro to nanoflow flow rate (nl / min) level.

  In recent years, when proteome analysis or the like is performed using a mass spectrometer, analysis is performed at an extreme flow rate of microliter (μl / min) to nanoliter (nl / min) level per minute.

  In the case of such an analysis, after separating the sample with a liquid chromatograph, the analysis is performed by ionizing with an electrospray ion source (ESI) of a mass spectrometer. The problem is that the sample diffuses due to the dead volume in the transfer path between the liquid chromatograph and the mass spectrometer. Therefore, in order to reduce this dead volume as much as possible, it is considered to use an integrated chip in which a separation column of a liquid chromatograph and an ESI nozzle are integrated. For example, the following has been reported.

  In JP 2003-151486 A (Patent Document 1), the tip of a quartz glass column has a pointed shape, an opening of 0.5 μm or less is provided, and a particle diameter of 0.5 μm to 5 μm is provided in this column. An example in which chemically bonded silica gel is used as a filler is described.

  However, in the case of using spherical particles as shown in Patent Document 1 as a liquid chromatograph column as a packing material, the resistance to fluid flow is large and the pressure loss is large. The problem that high pressure is required has been known. As an example for solving this problem, for example, in JP-A-6-265534 (Patent Document 2) and JP-A-2003-75420 (Patent Document 3), a column (monolithic column) using an inorganic porous material is used. )It is shown.

  The monolithic column is a rod-shaped porous body in which pores (macropores and mesopores) having different sizes are mixed. Macropores are about 2 microns (μm), whereas mesopores are tens of nanometers. Macropores facilitate solution flow and therefore, when used as a column, the pressure drop is a fraction of that of a normal column packed with a spherical packing, so it can be used at high flow rates. It has the following features. On the other hand, since the mesopore is several tens of nm, it helps to increase the specific surface area that is important for the column separation performance.

JP 2003-151486 A JP-A-6-265534 JP 2003-75420 A

  As an integrated chip that integrates the separation column used for LC / MS for analysis at the nanoflow level and the ESI nozzle, the general chip using granular fillers is not the above-mentioned pressure loss problem. In addition, the filler may leak out, or one particle of the filler may block the opening of the chip.

  In addition, when the above monolithic column is applied to an integrated chip, it is very difficult to manufacture a chip having a tip portion of several micrometers with good dimensional accuracy and high reproducibility. Furthermore, ionization efficiency, that is, signal intensity and sensitivity may be greatly affected by the size and shape of the tip.

  The present invention provides an integrated chip that is less likely to be clogged and can improve sensitivity and reproducibility in analysis at a micro to nanoflow flow rate, and a liquid chromatograph mass spectrometer using the same. It is intended.

In order to achieve the above object, the present invention is characterized by sharpening the tip of a capillary formed by filling a porous body in which pores of different sizes are mixed with each other by etching to expose the porous body. A conductive first coating film is formed on the first coating film, and the first coating film is grounded, and the exposed porous body is sharpened by FIB (Focusing Ion Beam) processing, and the porous portion is formed in the sharpened portion. An electrospray column-integrated chip manufacturing method comprising an opening at a position where a material is exposed and a conductive second coating film used as an electrode for applying a voltage of an electrospray ion source. There is .

  In accordance with the present invention, the possibility of clogging at the tip of the ESI chip, which often occurs in nanoelectrospray (ESI) mass spectrometers, is greatly reduced, and submicron-scale charged droplets are easily created, thereby providing a liquid It is possible to improve sensitivity and ensure reproducibility in a chromatograph-mass spectrometer.

  Further, by integrating the separation column and the ESI chip, it is possible to minimize the decrease in the column separation degree in the ESI section.

  Examples of the present invention will be described below.

  FIG. 1 shows a first embodiment of a column-integrated chip according to the present invention.

  As shown in FIG. 1, a monolithic column 1 that is an inorganic porous body is filled in a fused silica capillary 2 by a sol-gel reaction. Therefore, it is integrated with the fused silica capillary 2. The inner diameter of the fused silica capillary 2 is selected from 50 to 200 μm. This is because the diameter is necessary for filling the monolith column 1 and it is difficult to produce a diameter smaller than this.

  However, if the inner diameter size is as it is, it is difficult to produce a droplet (charged droplet) having a submicron level charge necessary for improving the sensitivity of the nanoflow ESI at the tip of the column. In nano flow level ESI, the smaller the tip opening diameter, the higher the sensitivity.

  Therefore, in this embodiment, in order to produce a tip portion of several micrometers, a column integrated chip is manufactured by the procedure shown in FIG.

  First, the tip of the fused silica capillary 2 having an inner diameter of 50 to 200 μm is sharpened to expose the column portion. However, when sharpening, in a processing method that is heated, the fused silica capillary 2 may melt and cover the column portion. Therefore, in this embodiment, the column portion is exposed by etching the tip portion of the fused silica capillary 2, for example, about several millimeters with hydrogen fluoride, and the outer diameter of the fused silica capillary 2 is exposed to the monolith column 1. Narrow until it is done or just before exposure.

  Thereafter, a conductive resin is applied to the fused silica capillary 2 to form a coating film, or a metal coating film is applied by plating. The coating film is grounded. This is for the subsequent FIB (Focusing Ion Beam) processing. FIB can be processed at a micrometer or less by atom bombardment by thinning ions into a beam and accelerating them. At this time, since the ions do not collide well if the potential of the object to be processed fluctuates, it is necessary to provide a conductive coating and further to ground.

  With FIB, it is possible to perform high-precision processing while observing the position with an electron microscope. Further, since processing is performed by atomic bombardment, heat is not applied to the member as in the case of a laser, and the member is not thermally denatured, so that the fused silica capillary 2 and the monolithic column 1 of different materials can be processed simultaneously. Furthermore, in FIB, automatic processing with high reproducibility is possible by NC (Numerical Control).

  In this embodiment, after forming the coating film, it is processed into a shape as shown in FIG. 1 (for example, a cone shape, a pyramid shape, a triangular pyramid shape, etc.) by FIB.

  After sufficiently thinning the tip, the coating film 3 is formed again except for the most advanced part. As the material of the coating, for example, a metal such as gold, a plastic resin mixed with carbon to maintain conductivity, or a conductive polymer film such as polyaniline is applied. In the case of metal, the forming method is plating, and in the case of conductive resin, the resin is applied. The vapor deposition method cannot be used because the monolith column 1 is porous. The coating film 3 is set so that the inner diameter of the uncoated portion is about several μm. Alternatively, once the entire tip portion is coated, the metal coating at the foremost portion may be removed by FIB processing so that the inner diameter becomes about several μm.

  In the present embodiment, even an existing monolithic column capillary having an inner diameter of 50 to 200 μm can realize a column-integrated chip having an opening of several μm that is optimal for a nanoflow level flow rate.

  3 to 6 show a second embodiment of the present invention. 3 to 5 are diagrams showing the integrated chip in the manufacturing process, and FIG. 6 is a flowchart of the manufacturing process.

  In this example, first, as shown in FIG. 3, a conductive coating film and a ground of this coating film are applied to a fused silica capillary 2 having an inner diameter of 50 to 200 μm filled with a monolith column 1 and subjected to FIB processing. Cut into a lotus shape. Next, as shown in FIG. 4, a coating film 3 is formed by plating or applying resin so that at least the cross-section of the tip portion is prevented from leaking from the monolith column 1. Thereafter, as shown in FIG. 5, the portion A at the tip of the lotus-shaped cut surface is cut by FIB processing. The cutting position at this time is such that the diameter of the cross section of the monolith column 1 that appears by cutting is about several μm.

  In this embodiment, compared with the first embodiment, the tip of the tip can be sharpened only by linear cutting, so that processing can be performed easily.

  7 and 8 show a third embodiment of the present invention.

  In this embodiment, the fused silica capillary 2 having an inner diameter of 50 to 200 μm, which is preliminarily packed with the monolith column 1, is used without being sharpened.

  In the example of FIG. 7, first, the tip end face of the fused silica capillary 2 is coated with a conductive resin so as to prevent leakage of the monolith column 1, the coating film 3 is applied, the coating film 3 is grounded, and then FIB A pinhole 5 having a diameter of several μm is opened by processing. In this embodiment, since the coating film 3 is first formed of conductive resin on the end face, the coating film 3 can be formed on the monolith column 1 which is a porous body, and the coating film 3 is It can be used as an electrode for applying an ESI voltage.

  FIG. 7 shows an example in which one pinall 5 is opened on the coating film 3, but a plurality of pinholes 5 may be formed on the monolith column 1. When a plurality of pinholes are formed, the sample can be sprayed more efficiently and pinhole clogging can be avoided.

In the example of FIG. 8, a coating film 3 made of a conductive resin having an opening formed in the monolith column 1 is applied to the tip of the fused silica capillary 2, and the tip made of a porous material is formed in the opening. The chip 4 is embedded in the tip of the monolith column 1. Since the sample eluted from the monolith column 1 is sprayed into the atmosphere through the tip 4, the sample can be sprayed more efficiently.

  In this embodiment, the sharpening process of the fused silica capillary 2 is not necessary, and the manufacture of the column integrated chip is facilitated.

  FIG. 9 shows a fourth embodiment of the present invention.

In this embodiment, a column packed with a conventional polymer or silica gel column filler 6 is combined with the column-integrated chip shown in FIG. As a manufacturing method, there are the following two methods.
(1) The monolith column 1 is formed and filled at the tip of the fused silica capillary 2, and then the above-mentioned column filler 6 is filled into the fused silica capillary 2 using a high-pressure pump. Thereafter, tip processing similar to that in Example 1 is performed.
(2) A fused silica capillary 2 filled with a monolithic column 1 in a state where the tip is not yet processed is connected to an empty fused silica capillary 2 using a connection union 7, and the column filler 6 is connected using a high-pressure pump. The fused silica capillary 2 is filled. Thereafter, tip processing similar to that in Example 1 is performed.

  In this example, a conventional column filler is packed afterwards on the upstream side of the monolith column 1, but since the back pressure of the monolith column is small, the packing shown in the above (1) and (2) is easy. Can be done. Moreover, since the monolith column 1 is packed at the tip, leakage of the column filler 6 in the subsequent stage can be prevented.

  According to the present embodiment, it is possible to provide a column in which two types of columns, an existing filler and a monolith column, are combined. As a result, two-dimensional LC and multi-dimensional LC can be realized with a single chip member without dead volume. For example, it can be used as an ion exchange column in the former stage and a reverse phase column in the latter stage. Therefore, a dedicated analysis column for multidimensional LC can be produced at low cost, and since it is combined with a monolithic column, it is possible to perform high throughput analysis at a low flow rate.

  FIG. 10 shows a liquid chromatograph / mass spectrometer as a typical application example of the column-integrated chip of the present invention.

  The eluents 9 and 10 are sent to the column-integrated tip 12 by the gradient function pump 8 while changing the composition ratio with time. The sample is introduced into the flow path by the sample injector 11, separated by the column integrated chip 12, and then sprayed as minute charged droplets 13. The charged droplet 13 is ionized by a voltage applied by the high voltage power supply 14 between the column integrated chip 12 and the ion intake orifice 15. The ionized charged droplet 13 enters the mass analyzer 16 from the ion intake orifice 15 and is subjected to mass analysis to be detected as a mass / charge ratio (m / z).

The detection sensitivity greatly depends on the size (submicron) of the charged droplet 13 formed at the forefront of the column integrated chip 12. By using the chip shown in each of the above embodiments as the column-integrated chip 12, it is possible to form the charged droplet 13 having an optimum size.

It is a figure which shows the 1st Example of a column integrated type | mold chip | tip. It is a flowchart which shows the manufacture process of the column integrated chip | tip of a 1st Example. It is a figure which shows the 2nd Example of a column integrated chip | tip. It is a figure which shows the 2nd Example of a column integrated chip | tip. It is a figure which shows the 2nd Example of a column integrated chip | tip. It is a flowchart which shows the manufacture process of the column integrated chip | tip of a 2nd Example. It is a figure which shows the 3rd Example of a column integrated chip | tip. It is a figure which shows the 3rd Example of a column integrated chip | tip. It is a figure which shows the 4th Example of a column integrated chip | tip. It is a figure which shows the structural example of LC / MS to which the column integrated chip of this invention is applied.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Monolith column, 2 ... Fused silica capillary, 3 ... Coating film, 4 ... Tip tip, 5 ... Pinhole, 6 ... Column filler, 7 ... Connection union, 8 ... Gradient pump, 9, 10 ... Eluent, DESCRIPTION OF SYMBOLS 11 ... Sample injector, 12 ... Column integrated chip, 13 ... Charged droplet, 14 ... High voltage power supply, 15 ... Ion intake orifice, 16 ... Mass spectrometry part.

Claims (1)

The porous body is exposed by sharpening the tip of the capillary formed by filling a porous body in which pores of different sizes are mixed, by etching treatment,
Forming a conductive first coating film on the capillary, and grounding the first coating film;
The exposed porous body is sharpened by FIB (Focusing Ion Beam) processing,
For the electrospray, wherein the sharpened portion is provided with an opening at a position where the porous body is exposed, and a conductive second coating film used as an electrode for applying a voltage of an electrospray ion source is applied. A method for manufacturing a column-integrated chip .

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