JP4370510B2 - Electrospray ionization nozzle for mass spectrometry - Google Patents

Description

The present invention relates to an improved technique related to mass spectrometry. Specifically, the present invention relates to a technique for improving a mass spectrometer and a mass spectrometry method in which sample molecules are introduced by spray ionization (hereinafter referred to as nanospray) by applying an electric field.

Mass spectrometer (hereinafter, referred to as "MS" (mass s pectrometer).) Is roughly composed of a "ion source" for ionization of the sample, m / ze is the ratio of ion mass / charge ( (analyzer) for separation according to m: mass, z: number of charges, e: unit charge), and “detection / recording unit” of separated ions.

An electrospray ionization technique generally known as “ESI” (abbreviation of electrospray ionization) is known as one of methods for ionizing and introducing sample molecules into an MS analyzer. In general, there is a spraying method in which high-pressure gas is sprayed onto a sample solution as a spray device. A high voltage is simultaneously applied between the solution and the sample inlet of the MS to superimpose electric charges on the droplets. One method that can be said to be a variation of this method is to use only the electric field applied between the sample and the sample inlet of the MS as the spray driving force to ionize the molecules in the sample solution using the nanoelectrospray or nanospray method. (Hereinafter referred to as nanospray method).

A common feature of this electrospray ionization technique is that micron-order liquids, in which a large number of solvent molecules are associated with polyprotonated molecules, are sprayed by applying high voltage to sample molecules that have been ionized in solution by acid or the like. A droplet (mist) is formed, nitrogen is blown in the vicinity of the MS sample introduction port, and then the solvent molecules are scattered and molecularized by entering the internal vacuum environment from the MS sample introduction port. This is a technique for guiding this to the analyzer. This technique is said to be useful for measurement of biopolymers such as peptides and proteins because the number of charges of ions generated in the polymer increases.

Here, various methods have been attempted for nano-preion ionization of sample molecules in the ion source of MS. One method will be described with reference to FIG. This is done by ejecting sample droplets little by little from the tip of the nozzle 1 to form a spray 2. The electric field is applied by applying either one of the positive and negative voltages to the internal sample solution 3 directly at the electrode 4 (changing polarity by positive charge ionization and negative charge ionization) or indirectly through a liquid junction. Part of the nozzle is made of metal, or more indirectly, but the periphery of the nozzle is coated with metal, a slight pressure (back pressure) is applied from the rear end of the nozzle, and the sample liquid leaks to the tip of the nozzle. Is applied and applied at the moment of contact with the metal coating surface, and one of the voltage ends applies an electric field to the metal portion 7 of the sample introduction part 6 of the MS main body 5, and the electric field and the driving force between them apply a charge. It is thought to fly in the form of microdroplets placed in large quantities. However, in the case of direct application, there is a problem that gas is generated by electrolysis on the surface of the metal serving as the electrode, and thus there is a problem that liquid flow and electrical conduction are hindered.

FIG. 1 is also a simplified view of the configuration of a conventional nanospray ionization technique, and reference numeral 1 represents the shape of a typical conventional nanospray nozzle. The nanospray nozzle 1 formed so that the tip has a pointed shape is generally formed by heating and stretching a quartz glass tube.

  The nanospray nozzle 1 is a constituent member of an ion source of the mass spectrometer 5, and applies a high voltage to the metal coated around the nozzle, and the tip of the nanospray nozzle 1 sprays the mass spectrometer 5. A fine droplet 2 containing sample molecules is sprayed toward the inlet 6. However, in principle, it is simple at first glance, but it is quite difficult to adopt this method to analyze it. First of all, stabilization of the spray is indispensable. I found that I had to solve various nozzle problems.

  For example, the thinner the tip, the smaller the spray droplets, and the ionization itself may be more efficient, but the longer the tip, the greater the interaction between the sample solution and the tube wall. The tension is dominant, the liquid flow is hindered, and the resistance when discharging only with an electric field is increased. As a result, the spray 2 was not stable, sometimes the liquid flow was interrupted, and subsequent measurement could not be performed. Filling and setting up a valuable sample once again into another new nanospray nozzle takes time, effort, loss of valuable sample, and the cost of a disposable consumable nozzle. There were a lot of problems.

  Furthermore, although there are some expensive nanospray chips available, the tip inner diameter d is about 10 to 15 μm at most, which is not suitable for forming fine droplets. Processing to the inner diameter of the opening is further costly, and it is also expensive to form a sharp tip shape with the diameter. Not suitable for disposable use as a consumable. Even with quartz glass, the inner diameter of the tip is about 1 μm or less, and the tapered tip is pressed against the wall of the MS machine, and the tip is folded to obtain an optimum diameter that is larger than that and has good spraying conditions. Set. Therefore, it is difficult to set certain conditions, and the cut surface is not flat. Also, the operation performed while looking through the magnifier was complicated. Furthermore, since it is required to bend the tip, a cut surface that is not coated with metal remains in the opening at the tip, and the sample solution does not exude so much as to cover the opening. An electrical contact with the coating cannot be obtained, which also leads to destabilization of the spray. Furthermore, the sample placed in the tip has a sample tube with an inner diameter of 1 mm or less and the tip of the sample is micron-order, so the sample solution does not go to the tip as it is. It is necessary to extrude the sample solution to the tip of the head, for example by pressurizing with. At that time, if the sample solution is visible, the operability is good. The one made of metal is not only the cost, but also the one that gas is generated by electrolysis on the inner surface and the liquid flow is interrupted, and the whole outer wall of the tube is coated with metal, but after putting the sample Another problem was that the position of the sample solution inside was not known and the operability was poor.

  Furthermore, because the tip is so thin that it cannot be seen, the tip may be broken even if you do not remember applying it well during experimental operation, and it is necessary to take measures to protect the tip during operation. In addition, the positional relationship between the nanospray nozzle and the MS main sample inlet and the orientation of each other are extremely important for setting conditions that are stable and contain a large number of ionized molecules. The analyzer holds the nanospray nozzle part on the XYZ fine movement stage, and expands from two directions (from the top and side) so that the positional relationship between the tip part and the MS main body sample introduction part appears to expand. It is configured to be captured and adjusted with a video camera. The operation is complicated, and the cost of the apparatus for that is great.

  In mass spectrometry, if a non-volatile salt or the like is dissolved in the sample solution, the sample inlet with a vacuum inside is clogged with salt from which the solvent has been deposited, or ionization of the molecule to be analyzed is hindered. Therefore, pretreatment operations such as desalting are essential. If this can be done in this nanospray nozzle, the operation becomes simple and it is possible to measure with a small amount of sample, but now it is desalted with another device or process, and the solution after that is nanospray nozzle It is often measured after filling.

In order to solve some of these problems, the electrospray has a porous bead or gold-plated porous bead that does not reduce the tip of the electrospray and engages the end face of the capillary itself having a substantially large outer diameter. Efficient spraying from many holes (see, for example, Patent Document 1) or electrospray tip is narrowed, the tip diameter is 0.5 μm or less, and 0.5 μm or more from the tip to the inside A column packing material for chromatography having a size of 5 μm or less and aimed at desalting and separation (for example, see Patent Document 2) has been proposed.
However, these are required for the spray nozzles and their use as described above, and are compatible with electrode coating and internal sample presence confirmation, efficient ionization, securing an appropriate flow rate of the sample in the nozzle and cost. There is no disclosure for issues such as coexistence.

Special table 2003-514501 JP2003-151486A

  An object of the present invention is to solve the above problems and to provide a nozzle for mass spectrometry used for stable mass spectrometry, particularly a spray nozzle preferable for nanospray method.

According to the present invention, when a sample is introduced into an area of the outer surface of the tube member that includes at least the vicinity of the opening at the tip by heating and stretching the tube member, the presence of the sample is present. The present invention provides a nozzle for mass spectrometry characterized in that a non-adhered portion that can be confirmed is left and a metal is coated.

According to the present invention, it is possible to obtain a nozzle that is stable, inexpensive, and has good operability.
In addition, according to the invention, molecular ionization for simple mass spectrometry can be achieved, and the nozzle tip is less likely to be accidentally bent during the preparatory operation of the experiment, and a nozzle that can be easily set in position can be obtained.
Furthermore, in other inventions in this, it is possible to obtain a nozzle that can perform various pretreatments and ionizations for mass spectrometry, such as pretreatment concentration and separation, or selective substance capture and enzyme digestion. .

Hereinafter, embodiments of the present invention will be described with reference to FIGS.
FIG. 2 is an explanatory view showing a manufacturing process of the nozzle according to the present invention.
First, as shown in (a), a pipe 8 having a wire 9 disposed therein is prepared. The tube 8 is a quartz glass tube, and the inner wire 9 is a thin quartz glass wire that is the same as the tube, and is attached to the inner wall of the tube 8 over the entire length of the tube. The material of the wire 9 is not necessarily the same as that of the tube 8, and any material that melts and stretches in the same way when the tube is heated and stretched may be used.
Next, as shown in (b), the tube 8 of (b), preferably the center, is heated with an annular heating and stretching heating element (for example, heater) 10, and the tube 8 and the wire 9 are melted at the melting temperature. At the same time, the tube material 8 is pulled in the longitudinal direction of the tube on both sides, for example, both ends of the heating and heating element (for example, a heater), and brought into a state as shown in (c). As a result, a portion 11 narrowed by primary stretching is formed in the central portion of the tube material 8, and a first tapered portion 8a is formed on both sides thereof.

Thereafter, the portion 11 that has become thinnest by primary stretching at the center of the tube 8 shown in (c) is heated by an annular secondary heating and heating element 12 in the same manner as the primary tube 8 and wire. 9 is heated above the melting temperature and stretched. In this secondary extension, the pipe material 8 is separated into two parts by extending more than the primary, and a second taper part 8b is formed at the tip of the first taper part 8a. At the tip of the tapered portion 8b, the tip opening 8c having an inner diameter of preferably 1 to 3 μm is preferably communicated with the external space. That is, by secondary extension, as shown in (e), the main body portion 8d having the same inner diameter as the original tube material, the first tapered portion 8a whose inner diameter is greatly reduced at the tip, and further the inner diameter at the tip. Is formed of a second taper portion 8b that gradually decreases, and two nozzle bodies 13 that are connected to the external space are formed by the tip openings 8c and 8e inside.

The wire 9 inside the nozzle body 13 is thin at the tapered portion, but is stretched in the same manner as the tube, continuously extends from the opening 8e to the tip opening 8c, and has an end facing the opening 8c.
In addition, the two tapered portions 8a and 8b have a tapered shape with a large diameter reduction rate in which the first tapered portion 8a has a large inclination as a whole, that is, the inner diameter is suddenly decreased. The tapered portion 8b is formed as a gentle tapered shape with a small decrease rate toward the tip opening 8c. The heating element 12 for secondary heating and stretching may be combined with the heating element 10 for primary heating and stretching described above.

The nanospray nozzle body 13 obtained in (e) of FIG. 2 is then coated with gold on the outer surface of the quartz glass by sputtering or vacuum deposition, and is a conductive part for applying voltage and applied to the sample. A metal coating portion 14 (the coating portion is indicated by hatching) that serves as an electrode for applying a voltage is formed.
The metal to be coated may be platinum other than gold, and vacuum coating may be used as a coating method.
The metal coating portion 14 is not formed on the entire outer surface of the nanospray nozzle body 13, but the inner sample in which the body portion 8d and at least the first taper portion 8a are not coated over substantially the entire length thereof. The metal coating non-adhesion part 15 which can see is formed. The metal coating non-adhering portion 15 is preferably formed on a part of the second tapered portion 8b if possible.

As shown in FIG. 4, the metal coating is performed over the entire outer surface in the vicinity of the tip opening 8c of the nanospray nozzle body 13 without forming the metal coating non-adhering portion 15, and particularly forms the tip opening 8c. The entire surface of the tip end face 16 of the annular nanospray nozzle is coated. Preferably, a part is further coated to the tip end face mouth inner side 18, and a voltage is applied when the sample comes into contact with the metal coated on the tip end face 16 or the like. The tip inner diameter 17 of the nanospray nozzle 13 has an inner diameter of 1 to 3 μm as described above, and the inner wire 9 is continuously arranged up to the nanospray nozzle tip inner surface 18 facing the opening 8c. Has been.

In the nozzle shown in FIGS. 2 to 4, as shown in FIG. 2, in order to improve the infiltration of the sample solution in the tapered capillary to the vicinity of the tip, the wire 9 is formed as a part of the tube member 8 inside. To make it easier for the liquid to reach the tip. In the manufacturing method, the wire 9 and the tube 8 are simultaneously heated and stretched at the portion where the wire 9 is disposed, so that one end is tapered and close to the opening at the tip as shown in FIGS. The inner wire 9 is extended and arranged. Further, as shown in FIG. 3, the nanospray nozzle 13 is made of a tube member 8 made of a transparent or translucent material so that the end surface of the inner sample solution can be seen, and a metal coating 14 is applied to the outer surface thereof. The nanospray nozzle is characterized by leaving a non-adhering portion 15 (for example, in the longitudinal direction) that can be confirmed when the solution is introduced. In addition, since the position of the sample solution at the narrow part of the tip is particularly important, it is as close as possible to the vicinity of the tip (the tip is to have conductivity around the entire circumference and to apply electric field to the solution as much as possible. By leaving the non-adhered portion 15 (to the vicinity of the tip) (for example, in the longitudinal direction), the visibility of the position of the internal sample solution was ensured and the operability was improved.
In addition, if the tip is thin and long, the surface tension between the sample solution and the tube wall dominates and the liquid flow is obstructed, increasing the resistance when discharging only with an electric field, and the liquid flow is interrupted. Spray is not stable. In order to solve this, the heating and stretching of the tube member and the wire is performed by at least two stages of heating (using the heating elements 10 and 12 in the figure) from the original large inner diameter as shown in FIG. Thus, as shown in FIG. 13, it has a two-step taper and a minimum diameter at the tip at a short stretch distance, so that the liquid flow of the sample solution is not easily disturbed by the surface tension with the tube wall.

  In addition, in order to save the troublesome operation of folding the tip part to the optimum tip inner diameter during use, the process shown in FIG. Then, in order to make it as shown in FIG. 3, metal coating was performed by vacuum deposition or sputtering. In this way, the manufacturing method is improved so that the metal coated on the outer surface can be metal-coated at least at the opening end surface 16 at the front end and partly inside the opening end surface 18 as shown in FIG. It is characterized in that if it reaches the mouth 17, it can be easily brought into contact with the conductive portion to realize a stable nanospray. Further, the adhesion of the metal to the outer surface of the nozzle is tapered as shown in 15 of FIG. 3 so that there is a non-attached portion so that the presence of the sample solution in the tapered portion can be confirmed when the sample is introduced. I devised and realized what is characterized by being performed on the outer surface of the part.

  FIG. 5 shows another embodiment. The sheath 19 protects the tip and the tapered portions 8a and 8b on the outside of the spray nozzle 13 and helps to set the nozzle in the analyzer for mass analysis. Is the one that is attached. The sheath 19 is made of a transparent or semi-transparent material made of plastic and has a cylindrical shape whose inner diameter is slightly larger than the outer cylinder of the nozzle, and a scale 20 is printed or stamped on the outside. The sheath 19 is fitted to the outer cylinder of the nozzle 13 so that the position of the sheath 19 can be adjusted by the tester's operation. The sheath 19 not only protects the tip even during the transportation of the product, but also protects the tip from breaking without being noticed during operations such as filling the spray nozzle with a sample solution. This is helpful when setting the nozzle on the analyzer. When setting the nozzle to the analyzer for mass analysis, as shown in Fig. 5 (a), it is considered optimal if the sample nozzle is located at some millimeters from the spray nozzle 13 and the MS main body sample inlet 6 and the tip. If so, the scale 20 on the outer surface of the sheath 19 is preset to its length, and the angle and its tip are positioned at the center of the sample inlet and stopped at the point of contact. You can easily set the distance and position. At the start of nanospraying, the sheath moves as shown in FIG. 5B, and an electric field is applied. Also in this case, there is an effect of preventing an electric shock such as accidentally touching with a hand. Above all, the expensive X-Y-Z fine movement stage currently attached to commercially available mass spectrometers and position adjustment equipment such as a magnifying video camera and monitor are no longer required, and the position can be set with a simple magnifying glass. It also features improved operability and cost.

  The shape and color of this sheath is free, and the tip part may be dyed in red or fluorescent color so that the position of the tip part can be seen well, and the tip tapered part is also sheathed. It may be tapered so that it fits (however, when the sheath is pressed against the nozzle, the tip of the nozzle comes out and nanospraying is possible). In addition, lines and dots may be engraved in the top, bottom, left, and right, so that the center of the MS sample inlet can be easily centered, and the tip is cut diagonally on two or four sides of the object. It may be dropped and devised such as vertical and horizontal positioning and smooth airflow during spraying. Various colors and shapes at the tip are conceivable depending on the purpose.

  FIG. 6 shows another embodiment. In the interior space of the nanospray nozzle 13 shown in FIGS. 3 and 4, a liquid chromatography resin, a sample pretreatment concentration resin, or a surface with a ligand, enzyme, protein or The resin 22 having the gene immobilized thereon is filled. These nozzles 13 are provided with a stopper member for a filler that can pass the sample liquid but cannot pass the filler 22 at least at the filler end portion at the nozzle tip, specifically, the second taper portion 8b. There is a need to. In FIG. 6A, for example, a glass mesh, a chemical fiber frit, a stainless steel filter member, or a hook-like member 21 is installed as the stopper member. 6B is provided with a plastic or ceramic porous member or a foamed porous member 23 that can pass the sample liquid but cannot pass the filler 22 near the tip of the stopper member. . The porous member and the foamed porous member 23 are preferably a porous resin for liquid chromatography. These stop members 21, 23 are formed to have an outer shape larger than the inner diameter of the nozzle tip opening, and stop before the nozzle 13 tip opening. By filling the inside with liquid chromatography resin, pretreatment resin or resin with immobilized ligand, enzyme, protein or gene on the surface, which is larger than the diameter of the spider or frit pores, only desalting and separation are possible. There is no high pressure for sample pretreatment concentration targeting analyte molecules, selective capture of proteins and genes, and selective enzyme digestion, even without using expensive devices such as ultra-trace metering pumps. Alone, mass spectrometry can be performed with nanospray while forming a natural liquid flow around the electric field drive.

Illustration of nanospray ionization Manufacturing method explanatory drawing of the nano spray nozzle main body which shows embodiment of this invention Diagram showing the entire nanospray nozzle Nano spray nozzle tip enlarged view Explanatory drawing of the nanospray nozzle sheath which is another embodiment of the present invention and how to use it Sectional drawing of the nano spray nozzle which is other embodiment of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Nano spray nozzle 2 Spray part by nano spray 3 Sample solution 4 Electric field application point 5 to sample solution Mass spectrometer main body 6 Mass spectrometer sample (ionization) introduction part 7 Mass spectrometer sample (ionization) introduction part Electric field application point 8 Tubing material 9 Wire material 10 Heating element for primary heating drawing (for example, heater)
DESCRIPTION OF SYMBOLS 11 Part thinned by primary extending | stretching 12 Heating element for secondary heating extending | stretching 13 Nano spray nozzle main body 14 Metal coating part 15 Metal coating non-adhering part 16 Nano spray nozzle front end surface (metal coating is carried out)
17 Nanospray nozzle tip inner diameter 18 Nanospray nozzle tip inner surface 19 Sheath 20 attached to nanospray nozzle 20 Distance setting scale attached to sheath 21 Saddle child-like member 22 Various resins 23 Saddle child-like resin

Claims (8)

    It is a part of a pipe member in which a wire is disposed, and the portion where the wire is disposed is heated and stretched together with the wire so that one end is tapered and close to the opening at the tip. The wire is configured to be extended, and a non-adherent portion is provided so that the presence of the sample can be confirmed when the sample is introduced into an area on the outer surface of the tube member including at least the vicinity of the opening at the tip. An ionization nozzle for mass spectrometry, characterized in that it is formed by coating a metal. 2. The electrospray ionization nozzle for mass spectrometry according to claim 1, wherein only the tube member or both the tube member and the wire are made of glass, and the heat stretching is performed at least twice. 3. The mass according to claim 1, wherein the metal attached to the outer surface is gold or platinum, the attachment is performed by sputtering or vapor deposition, and the metal is also attached to the opening end face of the tip. Electrospray ionization nozzle for analysis. The taper part has two taper parts with different diameter reduction rates formed by heating and stretching twice, and the taper part near the tip opening is configured to have a smaller rate of reduction than the other, and to the outer surface of the nozzle. claim deposition of metals, characterized in that have been made on the outer surface of the tapered portion such that there is non-adherent portion so there is ascertainable sample in tapered portion when a sample is introduced into 3 The electrospray ionization nozzle for mass spectrometry as described.       A tubular nozzle that ejects a sample for mass analysis from a small-diameter opening at the tip, a tubular member comprising a tubular portion and a tapered portion whose outer diameter decreases toward the tip opening, and the tubular portion inside the tubular portion And the wire member disposed over the vicinity of the opening of the tapered portion, the cylindrical portion of the outer surface of the tubular member, and the metal attached to the tapered portion, and the tapered portion has an inner diameter reduction rate toward the tip opening. The sample has two tapered parts with different thicknesses, and the metal is attached to the tapered part on the opening end surface of the tubular member. On the other hand, when the sample is introduced, the sample in the cylindrical part or the tapered part. An electrospray ionization nozzle for mass spectrometry, which is attached so that a non-attached part exists so that the presence of the water can be confirmed. The two taper parts are configured so that the rate of decrease of the taper part near the tip opening is smaller than the other, and at least the taper part is provided with a wire for improving the infiltration of the sample solution to the tip opening. The electrospray ionization nozzle for mass spectrometry according to claim 5 . The interior space is filled with liquid chromatography resin, solution sample pretreatment resin, or the surface is filled with ligand, enzyme, protein or resin immobilized resin, and at least the nozzle tip filling end is filled with liquid that can pass through. 7. An electrospray ionization nozzle for mass spectrometry according to claim 1 , further comprising a saddle-like member through which the agent cannot pass. The electrospray ionization nozzle for mass spectrometry according to claims 1 to 7, further comprising a sheath-like insulator that moves along the outer surface of the tube member.

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