DE102004048380A1 - Analysis of an element concentration of a sample, comprises irradiating the sample with the help of a laser ablation- inductive coupled plasma mass spectrometer - Google Patents

Abstract

The laser has a density of 109> W/cm2>, and surface diameter of a less than 100 mu m. The standard solutions are sputtered with a microspayer (3) into the plasma product by the laser ablation. The solution (less than mu l/min) is added by spraying pumps. An independent claim is also included for a device by using the above method.

Description

The The invention relates, on the one hand, to a method for the quantitative determination of elemental concentrations in solid samples and on the other hand for quantitative local analysis and distribution analysis. Further concerns the invention a for carrying out the the aforementioned method suitable device.

When Analysis method for the determination of trace elements down to the ng / g and sub-ng / g concentration range and lateral element distributions in the μm range are currently in mass spectrometry for direct ablation of the solid sample to be examined, various methods z. B. using focused laser beams applied. For example, the method of laser ablation is known - inductively coupled plasma mass spectrometry (LA-ICP-MS) in the trace and micro-local analysis. This method is disadvantageous, however able to directly determine quantitative elemental concentrations.

Other direct analysis methods for the determination of trace concentrations (e.g., GDMS glow discharge mass spectrometry or RFA - X-ray fluorescence analysis), are not sensitive on a regular basis enough or even unsuitable, quantitative analysis data of trace elements in sample materials to be tested or require them sample digestion of the solid state material (e.g. Mass spectrometry with inductively coupled plasma ion source - ICP-MS), what in particular for Ceramics is often difficult. In addition, these are analytical methods usually not for Distribution analyzes of non-conductive sample materials (including biological Samples such as tissue sections) due to charging effects on the Sample surface suitable and accordingly require special techniques to be developed.

In increasing dimensions become in the analytics laser, as for example the Nd YAG laser with one wavelength in the UV range (λ-266 nm or 213 nm). These lasers are often used for sample entry in high-detection ICP mass spectrometer (LA-ICP-MS: laser ablation inductively coupled plasma mass spectrometry) used. At present, Nd-YAG laser systems with a laser spot diameter of a few μm up to a few hundred μm For microlocal analysis commercially available (eg LSX 200, CETAC Technologies, Ohama, USA or UP 213, New WAVE Research, Fremont, USA).

The disadvantage is that the method of LA-ICP-MS does not directly provide quantitative analysis results. If the irradiated laser power density is more than 10 ⁹ W / cm ² , the sample material is ablated almost stoichiometrically. A stoichiometric ablation in the sense of this invention is understood to mean that the atomic composition of the vapor produced by the laser-solid-state interaction is identical to that of the laser-irradiated solid. This advantageously allows a simple semiquantitative analysis of the sample material if the concentration of an element is known (eg a matrix or trace element that can be used for internal standardization (interstandard element)), wherein the element concentrations in any sample materials with an error factor of 2 to 3 are determinable. At one of these irradiated laser power densities of more than 10 ⁹ W / cm ² , the quantification of the mass spectrometric analysis data is substantially simplified, since fractionation effects are usually minimized.

For many Questions in material development, materials research, in of microelectronics, in geology and in the life sciences However, quantitative analysis data required with high precision. Therefore it is of particular importance, appropriate quantification methods to develop. The different calibration strategies in the LA-ICP-MS have already been discussed in [1]. Particularly advantageous are on-line Methods of solution calibration, since they allow internal standardization and possible matrix and fractionation effects become. For all methods of solution calibration in LA-ICP-MS were so far the atomization the standard solution by means of a suitable atomizer [2, 3, 4] or microsprayers with desolvator [5] and the ablation of the solid sample to be examined separated from each other in a laser ablation chamber. Advantage of this Calibration with a monogas flow system compared to the dual gas flow system [6] is a better optimization of the experimental parameters and Consequently, lower detection limits, better accuracy and precision of Analysis results.

However, one problem of solution calibration in LA-ICP-MS, depending on the nebulizer, is the different element sensitivities of ICP-MS (eg, 3-5 orders of magnitude higher using an ultrasonic nebulizer) compared to the much lower element sensitivities in LA-ICP-MS However, these can be corrected via an internal standard element. Problems arise when the atomization of the standard solutions and the laser ablation of the solid sample are spatially separated, especially if there is no ideal mixture of the two aerosols in the laser ablation chamber. Ideally, therefore, a direct incorporation of a micro-atomizer into the laser ablation chamber, a direct atomization of the standard solution in the laser plasma [7] and a better Matri to ensure adaptation.

The The object of the invention is to provide a method available on the one hand, a simple quantitative determination of element concentrations in different sample materials (bulk analysis) allows and on the other hand a quantitative local and distributional analysis on surfaces of solid samples, in biological tissue samples, e.g. b. thin tissue sections and in with Gel electrophoresis separated protein spots in one- and two-dimensional Gels allowed. Another object of the invention is to provide a for execution of the method to provide suitable device.

The Objects of the invention are achieved by a method with the set of features according to the main claim, and by a device with the totality of features according to the independent claim. Advantageous versions of the method and the device can be found in each of them referred back Claims.

object the invention

The The invention relates to a method in which a special microspray in a laser ablation chamber is installed, wherein a stoichiometric Laser ablation of the investigated material is sought. there can in addition to any solid samples also advantageous to study biological materials. Under Biological materials are in particular materials in Cell organelles (eg mitochondria) in isolated protein spots in two-dimensional gels, biological tissue sections or biological To understand structures. The laser ablation of the biological material usually takes place in a laser ablation chamber with a cooled target holder. The laser ablation chamber is advantageous with an ICP-MS or also less sensitive ICP-OES (OES-optical emission spectrometry) coupled. The bombardment of the sample surface is done with photons of a Lasers, where a spatial resolution (Laserspotdurchmesser) on the surface of the sample of a few microns feasible are. In the wall of the laser ablation chamber becomes a special commercially available Microatomizer.

At a power density of the laser of at least 10 ⁹ W / cm ² , advantageously even up to 10 ¹⁰ W / cm ² on the surface of the sample, the ablation of the sample material in the laser ablation chamber is usually carried out stoichiometrically. The sample material thus ablated is then transported with a carrier gas, for example an argon stream, into the inductively coupled plasma (ICP) of a highly selective ICP mass spectrometer and ionized. Subsequently, the ions are separated according to their mass to charge ratio and sensitively detected. A determination of the elemental composition down to the ultratrace range and a distributional analysis of the examined sample material are possible with this method according to the invention. As an alternative detector system, for example, the ICP-OES is also considered.

During the measurement, a pulsed laser beam of the power density of at least 10 ⁹ W / cm ² , advantageously even up to 10 ¹⁰ W / cm ² with a spot diameter in a wide range of on the one hand 20- 50 microns on the other hand in the sub-micron on the sample surface Focused area. To quantify the LA-ICP-MS, defined standard solutions are atomized with a micro-atomizer into the plasma generated by laser ablation. Various calibration techniques can be used here.

• 1. The external calibration can be used if a blank of the same matrix is available. Here, a calibration line is created for each element by successively atomizing the standard solutions with defined element concentrations, while the blank is ablated with a focused laser beam under optimal experimental conditions. After establishing the calibration curve, the sample to be examined is ablated with unchanged experimental parameters, with the micro-atomizer a 2% HNO ₃ solution is introduced into the plasma. Only in this way can equivalent experimental conditions be guaranteed. To correct the different sensitivities of laser ablation ICP-MS and ICP-MS using the micro-atomizer, a suitable internal standard element (eg, a matrix element known concentration) is used.
• 2. If there is no blank, the standard addition method may be used be applied. Here, the sample to be analyzed with the laser ablated and then become the standard solutions systematically atomized with increasing concentration. From the obtained calibration line let yourself under consideration of different element sensitivities of both sample entry methods determine the element concentration of the analyte. Advantage of this Calibration strategy compared to external calibration is that this is an ideal matrix match.
• 3. However, ideal calibration in LA-ICP-MS can be achieved using isotope dilution analysis [8]. Here, the solution of a highly enriched isotope spikes in the Ma terial plasma of the ablated sample. Using on-line isotope dilution analysis, as isotope ratios are measured, potential plasma fluctuations or instrumental instabilities do not matter in the measurement.

The invention further relates to a device which is suitable for carrying out the aforementioned method. The device comprises a laser with a power density of at least 10 ⁹ W / cm ² on the surface of a sample to be analyzed, a laser ablation chamber, and a downstream mass spectrometer. To apply the on-line solution calibration in LA-ICP-MS, a micro-atomizer is directly installed in the laser ablation chamber. The sample supply of the standard solutions is defined via a high-precision syringe pump in the lower μL / min range.

Also the entire sample table with the laser ablation chamber is advantageous arranged on a 3-dimensional displacement unit, which also has a displacement resolution of at least 2 μm should have. this makes possible a quantitative elemental distribution analysis on solid samples or biological samples by LA-ICP-MS.

optional is additional a high-resolution microscope provided so that in addition to the spectroscopic analysis also a microscopic examination of the sample can be made.

By means of the laser ablation ICP mass spectrometry (LA-ICP-MS) according to the invention with on-line solution calibration, the simultaneous and direct quantitative elemental analysis on sample surfaces of a wide variety of solid-state samples can be made possible by means of a rapid scanning method. For the laser ablation ICP-MS of protein spots in 2D gels, a direct determination of phosphorus or even metal concentrations at a corresponding power density of 10 ⁹ W / cm ^{2 is} possible.

special Description part

following the object of the invention is explained in more detail with reference to a figure, without that the subject matter of the invention is limited thereby should. The figure shows: Experimental arrangement of the analysis method according to the invention with laser ablation chamber with a built-in micro-atomizer for on-line calibration of the LA-ICP-MS.

there mean in the figure:

1

laser ablation

2

Built microatomizer

3

Solid-state lasers (Nd-YAG) laser

4

frequency doubler (Second Harmonic Generation = SHG)

5

frequency tripler (Third Harmonic Generation = THG)

6

Frequenzverfünfacher (Quintuple Harmonic Generation logo CNRS logo INIST

QHG)

7

CCD camera

8th

zoom microscope

9

sample holder

10

cover

11

zoom system

12

focusing lens

13

Peltier cooling element

14

Mikromanipulatorversteller (x / y / z: 2 μm Resolution)

15

detection system (ICP-MS or ICP-OES)

When embodiment becomes a device according to the invention presented the following requirements of a quantitative Analysis procedure for LA-ICP-MS is sufficient: Development of a special cooled Ablation chamber with micro-atomizer for the direct quantitative determination of elemental concentrations in solid samples and of tissue sections. For biological samples, such as tissue sections directly To analyze, the sample holder should be in the laser ablation chamber (For example, with a Peltier element) are cooled.

• • Mikrozerstäuber zur definierten Zerstäubung von Standardlösungen zur Erstellung von Kalibriergeraden oder zur Zerstäubung hochangereicherter Isotopenspikes für die Isotopenverdünnungsanalyse.
• • Blitzlampengepumpter Nd-YAG Festkörperlaser in MOPA (Oszillator-Verstärker Anordnung)
• • Emission gepulster Strahlung mit Pulsbreite < 5 ns, Wiederholraten von bis zu 20 Hz und gausförmigen transversalen Strahlenprofil.
• • Frequenzkonversion durch harmonische Vervielfachung unter Ausnutzung nicht linearer Effekte in doppelbrechenden Kristallen
• • Gleichzeitige Generierung von gepulster Laserstrahlung der Wellenlänge 1064 nm, 532 nm und 213 nm. Je nach Applikation kann die jeweilige Strahlung entsprechender Wellenlänge mittels Kippspiegel zu- oder abgeschaltet werden.
• • Nachgeschalteter Strahlengang bestehend aus Abschwächer, Energiemessung, Strahlmanipulator, Wellenlängenseparatoren und Fokussieroptik.
• • Fokussieroptik bestehend aus Zoomteleskop und Objektivlinse, Strahlung der Wellenlänge 213 nm kann kleiner als 10 μm auf Probenoberfläche fokussiert werden.
• • Fokussieroptik für gleichzeitige Fokussierung der Strah lungen der Wellenlängen 1064 und 532 nm auf Nanospitze.
• • Echtzeit Probenbeobachtung mit Hilfe eines Zoommikroskops, einer CCD Kamera und einer Bildbearbeitungssoftware während des Ablationsprozesses (erforderliche Auflösung < 2 μm)
• • 3 dimensionale Probenpositionierung mit einer Auflösung von < 2 μm.
• • Einsatz eines zusätzlichen, stark vergrößernden optischen Mikroskops (< 500x) außerhalb der Strahlachse zur Feinpositionierung der Nanospitze.
• • Ablationskammer mit Eintrittsfenster für Laserstrahlung, Beobachtung und Mikromanipulatorversteller. Alle Eintrittsfenster sind abgedichtet.

The analysis system according to the invention is characterized in particular by a laser ablation system having the following elements and properties:

• • Microsprayers for the defined atomization of standard solutions for the creation of calibration lines or for the atomization of highly enriched isotope spikes for the isotope dilution analysis.
• • Flash lamp pumped Nd-YAG solid state laser in MOPA (oscillator amplifier arrangement)
• • Emission of pulsed radiation with pulse width <5 ns, repetition rates of up to 20 Hz and Gaussian transverse beam profile.
• • Frequency conversion through harmonic multiplication by utilizing nonlinear effects in birefringent crystals
• • Simultaneous generation of pulsed laser radiation of the wavelengths 1064 nm, 532 nm and 213 nm. Depending on the application, the respective radiation of the corresponding wavelength can be switched on or off by tilting mirrors.
• • Downstream beam path consisting of Attenuator, energy metering, beam manipulator, wavelength separators and focusing optics.
• • Focusing optics consisting of zoom telescope and objective lens, radiation of wavelength 213 nm can be focused smaller than 10 μm on sample surface.
• • Focusing optics for simultaneous focusing of the 1064 and 532 nm wavelengths on nanotipes.
• • Real-time sample observation using a zoom microscope, a CCD camera and image processing software during the ablation process (required resolution <2 μm)
• • 3-dimensional sample positioning with a resolution of <2 μm.
• • Use of an additional, magnifying optical microscope (<500x) outside the beam axis for fine positioning of the nanotip.
• • Ablation chamber with entrance window for laser radiation, observation and micromanipulator adjuster. All entrance windows are sealed.

• 1. Lösungskalibration der LA-ICP-MS unter Verwendung von Standardlösungen über externe Kalibration oder die Standardadditionsmethode [9]
• 2. Entwicklung eines neuartigen Verfahrens zur Isotopenverdünnungsanalyse unter Verwendung der Lösungskalibration: Hierbei erfolgt die Quantifizierung des S, Cu, Zn, Fe, Si oder anderer Elemente mit mindestens zwei Isotopen über die Zugabe des hochangereicherten Isotops in der Lösung nach Zerstäubung mit einem mikrokonzentrischen Zerstäuber in das Materialplasma, der direkt in Laserablationskammer eingebaut wurde. Man erhält zunächst ein konstantes Signal des zu messenden Isotopenverhältnisses de hochangereicherten Spikes. Anschließend wird die Probe ablatiert und die Isotopenmischung massenspektrometrisch analysiert. Aus der Änderung des Isotopenverhältnisses in der Mischung kann man über die Formel der Isotopenverdün nungsanalyse die Elementkonzentration bestimmen.

The quantification of the analytical results of the on-line solution calibration LA-ICP-MS according to the invention can be carried out via various quantification strategies, as listed below.

• 1. Solution calibration of LA-ICP-MS using standard solutions via external calibration or the standard addition method [9]
• 2. Development of a novel method for isotope dilution analysis using the solution calibration: Here, the quantification of S, Cu, Zn, Fe, Si or other elements with at least two isotopes via the addition of highly enriched isotope in the solution after atomization with a micro-concentric atomizer in the material plasma that was incorporated directly into the laser ablation chamber. First, a constant signal of the isotope ratio of the highly enriched spike to be measured is obtained. The sample is then ablated and the isotope mixture analyzed by mass spectrometry. From the change in the isotope ratio in the mixture, the element concentration can be determined by the formula of the isotope dilution analysis.

• • Quantitative Bestimmung von Spurenelementen in Festkörperproben (z. B. hochreinen Materialien) mittels on-line solution based LA-ICP-MS.
• • Quantitativen Mikrolokalanalyse an Proteinspots in 2D Gelen.
• • Quantitativen Verteilungsanalyse an Festkörperoberflächen (z. B. S-Layer, Nanocluster, Mikroarrays) an Interfaces oder an dünnen Schichten (Mikroelektronik, Materialforschung).
• • Quantitativen Verteilungsanalyse an biologischen (z. B. Baumringen), medizinischen Proben (Zähnen, Knochen, Haare u. ä.) oder an geologischen Proben (Einschlüsse, Inhomogenitäten).

The analytical method according to the invention is advantageously suitable in particular for the following application examples:

• • Quantitative determination of trace elements in solid samples (eg high-purity materials) using on-line solution based LA-ICP-MS.
• Quantitative microlocal analysis on protein spots in 2D gels.
• • Quantitative distribution analysis on solid surfaces (eg S-layers, nanoclusters, microarrays) on interfaces or on thin layers (microelectronics, materials research).
• • Quantitative distribution analysis on biological (eg tree rings), medical samples (teeth, bones, hair, etc.) or on geological samples (inclusions, inhomogeneities).

In cited literature:

• [1] J.S. Becker, "Application of ICP-MS and LA-ICP-MS in materials science ", Spectrochim, Acta B, 57 (2002) 1805);
• [2] "Ultrasonic nebulizer ": Pickhardt, C., Becker J.S., Dietze, H.-J., New calibration strategy of solution calibration in LA-ICP-MS for multielement trace analysis on geological samples ", Fresenius J. Anal. Chem. 368 (2000) 173-181;
• [3] Becker J.S., Pickhardt, C., Dietze H.-J., "Determination of trace elements in high purity platinum by laser ablation inductively plasma mass spectrometry using solution calibration ", Journal Analytical Atomic Spectroscopy 16 (2001) 603-606;
• [4] Becker, J.S., Boulyga, S.F., Pickhardt, C., Becker, J. Su. Damoc, N.-E., Przybylski, M., "Structural Identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron inductively coupled plasma mass spectrometry ", Int. J. Mass Spectrom. 228 (2003) 985-997;
• [5] Aridus, Boulyga, S. F., Pickhardt, C., Becker, J.S., "New approach to solution-based calibration laser ablation ICP-MS of trace elements in metals and reduction of fractionation effects ", Atomic Spectroscopy 25 (2004) 53-63);
• [6] J.J. Leach, L.A. Allen, D.B. Aschliman, R.S. Houk, "Calibration in laser ablation inductively coupled plasma mass spectrometry using standard addition with dried solution aerosol ", Anal. Chem. 71 (1999) 440-445);
• [7] "Material plasma the ablated solid sample ", J.S. Becker, D. Tenzler, Fresenius J. Anal. Chem. 370 (2001) 637);
• [8] J.S. Becker, C. Pickhardt, W. Pompe, Int. J. Mass Spectrom. 237 (2004) 13);
• [9] J.S. Becker, Spectrochim. Acta B57 (2002) 1805);

Claims (7)

Method for the analysis of elemental concentrations of a sample by means of laser ablation - inductively coupled plasma mass spectrometry (LA-ICP-MS), characterized in that the irradiated laser power density is at least 10 ⁹ W / cm ² and the laser has a spot diameter on the surface of the sample of less than 100 microns. The method of claim 1, wherein the defined standard solutions with a micro-atomizer be atomized into the plasma generated by the laser ablation. Method according to one of the preceding claims 1 to 2, where the standard solutions about spray pumps in the lower μL / min be dosed. Method according to one of the preceding claims 1 to 3, wherein a laser power density is set at least 10 ⁹ W / cm ² . Apparatus for analyzing elemental concentrations of a sample by laser ablation - inductively coupled plasma mass spectrometry (LA-ICP-MS) comprising a laser having a power density of at least 10 ⁹ W / cm ² on the surface of a sample to be analyzed, a laser ablation chamber and a downstream one Detector system, characterized in that in the laser ablation chamber a micro atomizer is seconded. Apparatus according to the preceding claim 5, wherein the micro atomizer is arranged in the wall of the laser ablation chamber. Device according to one of the preceding claims 5 to 6, with a mass spectrometer as a detector system.