JP2009186218A - Method for measuring mass spectrum of liquid chromatograph eluate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method that enables acquisition of a mass spectrum of a component eluted from a liquid chromatograph apparatus, and accurately and rapidly analyze a sample mixture even when an apolar solvent is used for GPC, normal phase HPLC or the like. <P>SOLUTION: The method for measuring a mass spectrum of a liquid chromatograph eluate comprises: preparing a target 2 having a surface on which an activating substance 1, which enables to obtain a mass spectrum by laser radiation to a sample, is entirely or linearly distributed; applying the sample solution eluted from a liquid chromatograph apparatus 3 to the target 2 while linearly moving the sample solution; measuring by an MALDI-TOF-MS apparatus 7 a mass spectrum of the entire surface of the target 2 or the part on which the sample solution has been linearly applied; and acquiring an image of two-dimensional distribution of an ion having a specific mass number. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring a mass spectrum of a liquid chromatograph eluate, and in particular, an analysis capable of obtaining a mass spectrum of each separated component even when a nonpolar solvent is used in GPC, normal phase HPLC or the like. It is about the method.

  In the fields of clinical, biochemical, chemical industry, etc., mass spectrometers are frequently used to perform qualitative or quantitative analysis on samples such as proteins and additives. The mass spectrometer performs analysis from a mass spectrum obtained by performing physical filtering using the mass and charge number of ions generated by applying energy to a sample. A time-of-flight mass spectrometer (TOFMS: Time of Flight Mass Spectrometry) that performs qualitative / quantitative analysis using the time from the head to the detector is known.

  In these analysis methods, especially when analyzing samples with large molecular weight such as biological materials such as proteins and polymers, the sample is mixed with an organic compound called a matrix and then dropped and dried on the sample plate. A MALDI (Matrix Assisted Laser Desorption / Ionization) method is generally known in which a sample is desorbed and ionized to perform mass spectrometry by irradiating it with an energy beam.

  On the other hand, in order to obtain the mass spectrum of the eluted component from the liquid chromatograph, it is common to use an LC-MS device equipped with an ESI ion source, but GPC using a nonpolar solvent or normal phase measurement It is also difficult to measure a polymer having a large molecular weight. Therefore, in order to obtain the mass spectrum of the eluted component from the liquid chromatograph, the fraction of the component eluted from the liquid chromatograph is collected in batches. For example, the fraction is combined with the matrix agent on the target of MALDI-TOF-MS. It was necessary to apply and measure mass spectra of many components one by one.

  By the way, although the MALDI method is highly sensitive as an analysis method, since the matrix itself is also decomposed and ionized, the matrix agent becomes a background noise in a low molecular weight region. Therefore, the MALDI method is not suitable for analyzing a sample whose separation target is a low molecular compound by the liquid chromatography method.

  On the other hand, recently, a technique for ionizing a sample without adding a matrix to the sample has been devised and put into practical use. A typical example is the DIOS (Desorption / Ionization on (porous) Silicon) method. This ionization method is a method for imparting the ionization ability of a sample to the sample plate itself, and by performing anodizing treatment (anodizing treatment: a kind of electrochemical anodizing method) on the surface of the Si substrate. A sample plate formed with a sample coating portion composed of a porous portion of submicron order is used as a sample plate. The name of DIOS originates from Si as a base material, but it is reported that the same effect can be obtained by other matrix-free ionization methods such as SALDI method using a material other than Si as a base material. ing. However, no attempt has been made to rapidly analyze a sample mixture using these matrix-free ionization methods.

Shoji Okuno, Yukiyasu Shimozen, Yoshinao Wada, Ryuichi Arakawa, “Application of Laser Desorption / Ionization Mass Spectrometry Using Porous Silicon to Synthetic Polymers”, Analytical Chemistry, 2005, Vol. 54, No. 6, p. 439-447

  Under such circumstances, the object of the present invention is to obtain a mass spectrum of components eluted from a liquid chromatograph apparatus even when a nonpolar solvent is used in GPC, normal phase HPLC, etc. It is an object of the present invention to provide a method capable of accurately and quickly performing the above.

  As a result of intensive studies to achieve the above object, the present inventor applied the eluate from the liquid chromatograph device to the target used in the matrix free ionization method, and has a specific mass number with imaging software. By obtaining a two-dimensional image of ions, it was found that the sample mixture can be analyzed accurately and quickly, and the present invention has been completed.

That is, the method for measuring the mass spectrum of the liquid chromatograph eluate of the present invention comprises:
Prepare a target with an active substance distributed over the entire surface or linearly, which makes it possible to obtain a mass spectrum by laser irradiation of the sample,
Apply the sample solution eluting from the liquid chromatograph to the target while moving it linearly,
Measure the mass spectrum of the entire surface of the target with the MALDI-TOF-MS apparatus or the mass spectrum of the portion where the sample solution is applied linearly,
An image of a two-dimensional distribution of ions having a specific mass number is obtained.

  In a preferred embodiment of the mass spectrum measurement method of the present invention, the activating substance that enables obtaining a mass spectrum by laser irradiation of the sample is a porous substance. Here, the porous material is preferably porous silicon.

  In another preferred embodiment of the method for measuring a mass spectrum of the present invention, the activating substance capable of obtaining a mass spectrum by laser irradiation of the sample is a nanoparticle and / or an aggregate of nanoparticles. Here, the nanoparticles are preferably a metal and / or an inorganic substance. The metal is more preferably platinum and / or gold, and the inorganic substance is more preferably zinc oxide and / or titanium oxide, and germanium nanodots grown on a silicon substrate.

  In another preferred embodiment of the method for measuring mass spectrum of the present invention, the activating substance capable of obtaining a mass spectrum by laser irradiation of the sample is an organic substance.

  In another preferred embodiment of the mass spectrum measurement method of the present invention, the sample solution eluted from the liquid chromatograph apparatus is applied to the target while moving the target placed on the XY stage.

  In another preferred embodiment of the mass spectrum measurement method of the present invention, the MALDI-TOF-MS apparatus is a time-of-flight mass spectrometer that obtains a mass spectrum by laser irradiation of a target coated with a sample.

  According to the present invention, even when a nonpolar solvent is used in GPC, normal phase HPLC, or the like, a rapid and detailed analysis based on a mass spectrum of a component eluted from a liquid chromatograph is possible.

  Hereinafter, the measurement method of the present invention will be described in detail with reference to the drawings. FIG. 1 is a schematic diagram of the measurement method of the present invention. In the measurement method of the present invention, first, a target 2 is prepared in which an activating substance 1 capable of obtaining a mass spectrum by laser irradiation of a sample is distributed over the entire surface or linearly. The activating substance is an ionizing active substance that enables ionization of a sample by laser irradiation.

  Here, the activating substance 1 that makes it possible to obtain a mass spectrum by laser irradiation of a sample is preferably a porous substance, and particularly preferably porous silicon. The pore diameter of the porous material used is preferably in the range of 1000 to 1 nm.

  Moreover, as the activating substance 1 that makes it possible to obtain a mass spectrum by laser irradiation of a sample, nanoparticles and aggregates of nanoparticles are also preferable. Here, as a nanoparticle, a metal and an inorganic substance are preferable. In addition, noble metals such as platinum and gold are preferable as the metal, and inorganic oxides such as zinc oxide and titanium oxide are preferable as the inorganic substance. The particle diameter of the nanoparticles is preferably in the range of 0.1 to 100 nm, and the size of the aggregate of nanoparticles is preferably in the range of 1 to 10000 nm.

  Examples of the activating substance 1 that enables obtaining a mass spectrum by laser irradiation of a sample include germanium nanodots grown on a silicon substrate, and the size of the nanodots is preferably 1 to 100 nm.

  The target 2 in which the activation material 1 is distributed over the entire surface or linearly is formed by etching the activation material, depositing the activation material on an existing target such as stainless steel, sputtering, or chemically treating the target 2. Can be prepared. Further, the pitch width of the activated substance 1 distributed linearly is not particularly limited, but a range of 1 to 10 mm is preferable.

  Next, in the measurement method of the present invention, the sample solution eluted from the liquid chromatograph apparatus 3 is applied to the target 2 prepared as described above while moving linearly. Here, the sample solution eluted from the liquid chromatograph is applied to the portion where the activated substance exists on the target. The liquid chromatograph 3 is not particularly limited, and usually includes a column 4 and a pump (not shown), and utilizes the difference in the retention time of each component in the sample when the sample passes through the column. To separate each component in the sample. Further, the operation conditions of the liquid chromatograph device 3, the column to be used, the type of the eluent, and the like are not particularly limited, and can be appropriately selected according to the analysis target.

  The sample solution eluted from the liquid chromatograph device 3 may be applied to a desired position on the target 2 while moving the humidified spray nozzle portion 5 linearly, or while fixing the nozzle portion 5. The target 2 mounted on the XY stage 6 may be applied to a desired position on the target 2 while moving. Further, if necessary, an ionizing agent such as sodium trifluoroacetate may be simultaneously applied to the target. Here, the pitch width of the linearly applied sample solution is not particularly limited, but is preferably in the range of 1 to 10 mm.

  Next, in the measuring method of the present invention, the MALDI-TOF-MS apparatus 7 measures the mass spectrum of the entire surface of the target 2 or the mass spectrum of the portion where the sample solution is applied in a linear form, and the specific mass number. An image of a two-dimensional distribution of ions having Here, as the MALDI-TOF-MS apparatus, a time-of-flight mass spectrometer that obtains a mass spectrum by laser irradiation of a target coated with a sample is preferable. The time-of-flight mass spectrometer is an analyzer that performs qualitative / quantitative analysis using the time required for product ions to reach a detector from an ion generator. Note that the laser irradiation conditions are not particularly limited, and can be appropriately adjusted according to the analysis target.

  The target 2 coated with the sample solution as described above is introduced into the MALDI-TOF-MS apparatus 7, and the mass spectrum of the entire surface of the target or the mass spectrum of the portion where the sample solution is coated linearly is slightly changed. A large number of ions are automatically measured while being moved, and an image of a two-dimensional distribution of ions with a specific mass is reconstructed with computer software for imaging, enabling rapid analysis by mass spectrum of each component separated by liquid chromatography. Identification becomes possible. Further, a more detailed structural analysis can be performed by the MS / MS spectrum of the separated components.

  The analysis target of the measurement method of the present invention described above is not particularly limited. For example, synthetic polymers and natural polymers having a molecular weight distribution of 100 to 10,000, additives for polymers such as antioxidants, sugars , Natural products such as proteins and peptides.

It is the schematic of the measuring method of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Activated substance 2 Target 3 Liquid chromatograph apparatus 4 Column 5 Nozzle part 6 XY stage 7 MALDI-TOF-MS apparatus

Claims (10)

Prepare a target with an active substance distributed over the entire surface or linearly, which makes it possible to obtain a mass spectrum by laser irradiation of the sample,
Apply the sample solution eluting from the liquid chromatograph to the target while moving it linearly,
Measure the mass spectrum of the entire surface of the target with the MALDI-TOF-MS apparatus or the mass spectrum of the portion where the sample solution is applied in a line, and obtain an image of a two-dimensional distribution of ions having a specific mass number. A method for measuring a mass spectrum of a liquid chromatograph eluate, which is characterized.   The method for measuring a mass spectrum of a liquid chromatograph eluate according to claim 1, wherein the activating substance capable of obtaining a mass spectrum by laser irradiation of the sample is a porous substance.   The method for measuring a mass spectrum of a liquid chromatograph eluate according to claim 2, wherein the porous substance is porous silicon.   The mass of the liquid chromatograph eluate according to claim 1, wherein the activating substance capable of obtaining a mass spectrum by laser irradiation of the sample is a nanoparticle and / or an aggregate of nanoparticles. Spectrum measurement method.   5. The method for measuring a mass spectrum of a liquid chromatograph eluate according to claim 4, wherein the nanoparticles are a metal and / or an inorganic substance.   The method of measuring a mass spectrum of a liquid chromatograph eluate according to claim 5, wherein the metal is platinum and / or gold.   The method for measuring a mass spectrum of a liquid chromatograph eluate according to claim 5, wherein the inorganic substance is zinc oxide and / or titanium oxide.   The mass spectrum of the liquid chromatograph eluate according to claim 1, wherein the activating substance capable of obtaining a mass spectrum by laser irradiation of the sample is germanium nanodots grown on a silicon substrate. Measuring method.   The method for measuring a mass spectrum of a liquid chromatograph eluate according to claim 1, wherein the sample solution eluted from the liquid chromatograph is applied to the target while moving the target placed on the XY stage.   The mass spectrum of the liquid chromatograph eluate according to claim 1, wherein the MALDI-TOF-MS apparatus is a time-of-flight mass spectrometer that obtains a mass spectrum by laser irradiation of a target coated with a sample. Measuring method.

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