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2.7 X-RAY FLUORESCENCE SPECTROMETRY

2.7 X-RAY FLUORESCENCE SPECTROMETRY

      Wavelength dispersive X-ray fluorescence spectrometry is a procedure that uses the measurement of the intensity of the fluorescent radiation emitted by an element having an atomic weight between 11 and 92 excited by a continuous primary X-ray radiation. The intensity of the fluorescence produced by a given element depends on the concentration of this element in the sample but also on the absorption by the matrix of the incident and fluorescent radiation. At trace levels, where the calibration curve is linear, the intensity of the fluorescent radiation emitted by an element in a given matrix, at a given wavelength, is proportional to the concentration of this element and inversely proportional to the mass absorption coefficient of the matrix at this wavelength.

METHOD Set and use the instrument in accordance with the instructions given by the manufacturer. Liquid samples are placed directly in the instrument; solid samples are first compressed into pellets, sometimes after mixing with a suitable binder.

      To determine the concentration of an element in a sample, it is necessary to measure the net impulse rate produced by one or several standard preparations containing known amounts of this element in given matrices and to calculate or measure the weight absorption coefficient of the matrix of the sample being analyzed.

CALIBRATION From a calibration solution or a series of dilutions of the element to be analyzed in various matrices, determine the slope of the calibration curve, b0, from the following equation:

WEIGHT ABSORPTION COEFFICIENT OF THE MATRIX OF THE SAMPLE If the empirical formula of the sample being analyzed is known, calculate its weight absorption coefficient from the known elemental composition and the tabulated elemental weight absorption coefficients. If the elemental composition is unknown, determine the weight absorption coefficient of the sample matrix by measuring the intensity of the scattered X-radiation IU (Compton scattering) from the following equation: 

DETERMINATION OF THE NET IMPULSE RATE OF THE ELEMENT TO BE DETERMINED IN THE SAMPLE Calculate the net impulse rate of the element being determined from the measured intensity of the fluorescence line and the intensity of the background line(s), allowing for any tube contaminants present.

CALCULATION OF THE TRACE CONTENT If the concentration of the element is in the linear part of the calibration curve, it can be calculated using the following equation:

2.8 MASS SPECTROMETRY

      Mass spectrometry is based on the direct measurement of the ratio of the mass to the number of positive or negative elementary charges of ions (m/z ) in the gas phase obtained from the substance being analyzed. This ratio is expressed in atomic mass units (1 a.m.u = one twelfth the mass of 12C) or in daltons (1 Da = the mass of the hydrogen atom).

      The ions, produced in the ion source of the apparatus, are accelerated and then separated by the analyzer before reaching the detector. All of these operations take place in a chamber where a pumping system maintains a vacuum of 10–3 to 10–6 Pa.

      The resulting spectrum shows the relative abundance of the various ionic species present as a function of m/z. The signal corresponding to an ion will be represented by several peaks corresponding to the statistical distribution of the various isotopes of that ion. This pattern is called the isotopic profile and (at least for small molecules) the peak representing the most abundant isotopes for each atom is called the monoisotopic peak.

      Information obtained in mass spectrometry is essentially qualitative (determination of the molecular mass, information on the structure from the fragments observed) or quantitative (using internal or external standards) with limits of detection ranging from the picomole to the femtomole.

Introduction of the Sample

      The very first step of an analysis is the introduction of the sample into the apparatus without overly disturbing the vacuum. In a common method, called direct liquid introduction, the sample is placed on the end of a cylindrical rod (in a quartz crucible, on a filament or on a metal surface). This rod is introduced into the spectrometer after passing through a vacuum lock where a primary intermediate vacuum is maintained between atmospheric pressure and the secondary vacuum of the apparatus.

      Other introduction systems allow the components of a mixture to be analyzed as they are separated by an appropriate apparatus connected to the mass spectrometer.

      GAS CHROMATOGRAPHY/MASS SPECTROMETRY The use of suitable columns (capillary or semi-capillary) allows the end of the column to be introduced directly into the source of the apparatus without using a separator.

      LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY This combination is particularly useful for the analysis of polar compounds, which are insufficiently volatiole or too heat-labile to be analyzed by gas chromatography coupled with mass spectrometry. This method is complicated by the difficulty of obtaining ions in the gas phase from a liquid phase, which requires very special interfaces such as:

      (1) DIRECT LIQUID INTRODUCTION : the mobile phase is nebulized, and the solvent is evaporated in front of the ion source of the apparatus;

      (2) PARTICLE-BEAM INTERFACE : the mobile phase, which may flow at a rate of up to 0.6 ml per minute, is nebulized in a desolvation chamber such that only the analytes, in neutral form, reach the ion source of the apparatus; this technique is used for compounds of relatively low polarity with molecular masses of less than 1000 Da;

      (3) MOVING-BELT INTERFACE : the mobile phase, which may flow at a rate of up to 1 ml per minute; is applied to the surface of a moving belt; after the solvent evaporates, the components to be analyzed are successively carried to the ion source of the apparatus where they are ionized; this technique is rather poorly suited to very polar or heat-labile compounds.

      Other types of coupling (electrospray, thermospray, atmospheric-pressure chemical ionization) are considered to be ionization techniques in their own right and are described in the section on modes of ionization.

SUPERCRITICAL FLUID CHROMATOGRAPHY/MASS SPECTROMETRY The mobile phase, usually consisting of supercritical carbon dioxide enters the gas state after passing a heated restrictor between the column and the ion source.

CAPILLARY ELECTROPHORESIS/MASS SPECTROMETRY The eluent is introduced into the ion source, in some cases after adding another solvent so that flow rates of the order of a few microlitres per minute can be attained. This technique is limited by the small quantities of sample introduced and the need to use volatile buffers.

Modes of Ionization

      ELECTRON IMPACT The sample, in the gas state, is ionized by a beam of electrons whose energy (usually 70 eV) is more than the ionization energy of the sample. In addition to the molecular ion M+ , fragments characteristic of the molecular structure are observed. This technique is limited mainly by the need to vaporize the sample. This makes it unsuited to polar, heat-labile or high molecular mass compounds. Electron impact is compatible with the coupling of gas chromatography to mass spectrometry and sometimes with the use of liquid chromatography.

      CHEMICAL IONIZATION This type of ionization involves a reagent gas such as methane, ammonia, nitrogen oxide, nitrogen dioxide or oxygen. The spectrum is characterized by ions of the (M + H)+ or (M – H) types, or adduct ions formed from the analyte and the gas used. Fewer fragments are produced than with electron impact. A variant of this technique is used when the substance is heat-labile: the sample, applied to a filament, is very rapidly vaporized by the Joule-Thomson effect (desorption chemical ionization).

FAST-ATOM BOMBARDMENT (FAB) OR FAST-ION BOMBARDMENT IONIZATION (LIQUID SECONDARY-ION MASS SPECTROMETRY, LSIMS) The sample, dissolved in a viscous matrix such as glycerol, is applied to a metal surface and ionized by a beam of neutral atoms such as argon or xenon or high-kinetic-energy caesium ions. Ions of the (M + H)+ or (M – H) types or adduct ions formed from the matrix or the sample are produced. This type of ionization, well suited to polar and heatlabile compounds, allows molecular masses of up to 10,000 Da to be obtained. The technique can be combined with liquid chromatography by adding 1 to 2 per cent of glycerol to the mobile phase; however, the flow rates must be very low (a few microlitres per minute). These ionization techniques also allow thinlayer chromatography plates to be analyzed by applying a thin layer of matrix to the surface of these plates.  

FIELD DESORPTION AND FIELD IONIZATION The sample is vaporized near a tungsten filament covered with microneedles (field ionization) or applied to this filament (field desorption). A voltage of about 10 kV, applied between this filament and a counter-electrode, ionizes the sample. These two techniques mainly produce molecular ions M+ and (M + H)+ ions and are used for low polarity and/or heat-labile compounds.

MATRIX-ASSISTED LASER DESORPTION IONIZATION (MALDI) The sample, in a suitable matrix and deposited on a metal support, is ionized by a pulsed laser beam whose wavelength may range from UV to IR (impulses lasting from a picosecond to a few nanoseconds). This mode of ionization plays an essential role in the analysis of very high molecular mass compounds (more than 100,000 Da) but is limited to time-of-flight analyzers (see below).

ELECTROSPRAY This mode of ionization is carried out at atmospheric pressure. The samples, in solutions, are introduced into the source through a capillary tube, the end of which has a potential of the order of 5 kV. A gas can be used to facilitate nebulization. Desolvation of the resulting microdroplets produces singly or multiply charged ions in the gas phase. The flow rates vary from a few microlitres per minute to 1 ml per minute. This technique is suited to polar compounds and to the investigation of biomolecules with molecular masses of up to 100,000 Da. It can be coupled to liquid chromatography or capillary electrophoresis. 

ATMOSPHERIC-PRESSURE CHEMICAL IONIZATION (APCI) Ionization is carried out at the atmospheric pressure by the action of an electrode maintained at a potential of several kilovolts and placed in the path of the mobile phase, which is nebulized both by thermal effects and by the use of a stream of nitrogen. The resulting ions carry a single charge and are of the (M + H)+ type in the positive mode and of the (M – H) types in the negative mode. The high flow rates that can be used with this mode of ionization (up to 2 ml per min) make this an ideal technique for coupling to liquid chromatography

THERMOSPRAY The sample, in the mobile phase consisting of water and organic modifiers and containing a volatile electrolyte (generally ammonium acetate), is introduced in nebulized form after having passed through a metal capillary tube at a controlled temperature. Acceptable flow rates are of the order of 1 ml per minute to 2 ml per minute. The ions of the electrolyte ionize the compounds to be analyzed. This ionization process may be replaced or enhanced by an electrical discharge of a bout 800 volts, notably when the solvents are entirely organic. This technique is compatible with the used of liquid chromatography coupled with mass spectrometry.

Analyzers

      Differences in the performance of analyzers depend mainly on two parameters:

      (1) the range over which m/z ratios can be measured, i.e. the mass range;

      (2) their resolving power characterized by the ability to separate two ions of equal intensity with m/z ratios differing by ΔM, and whose overlap is expressed as a give percentage of valley definition; for example, a resolving power (M/ΔM) of 1000 with 10 per cent valley definition allows the separation of m/z ratios of 1000 and 1001 with the intensity returning to 10 per cent a above baseline. However, the resolving power may in some cases (time-of-flight analyzers, quadrupoles, iontrap analyzers) be defined as the ratio between the molecular mass and peak width at half height (50 per cent valley definition).

MAGNETIC AND ELECTROSTATIC ANALYZERS The ions produced in the ion source are accelerated by a voltage V, and focused towards a magnetic analyzer (magnetic field B) or an electrostatic analyzer (electrostatic field E), depending on the configuration of the instrument. They follow a trajectory of radius r according to Laplace’s law:

      Two types of scans can be used to collect and measure the various ions produced by the ion source: a scan of B holding V fixed or a scan of V with constant B. The magnetic analyzer is usually followed by an electric sector that acts as a kinetic energy filter and allows the resolving power of the instrument to be increased appreciably. The maximum resolving power of such an instrument (double sector) ranges from 10,000 to 150,000 and in most cases allows the value of m/z ratios to be calculated accurately enough to determine the elemental composition of the corresponding ions. For monocharged ions, the mass range is from 2000 Da to 15,000 Da. Some ions may decompose spontaneously (metastable transitions) or by colliding with a gas (collision-activated dissociation, CAD) in field-free regions between the ion source and the detector. Examination of these decompostitions is very useful for the determination of the structure as well as the characterization of a specific compound in a mixture and involves tandem mass spectrometry. There are many such techniques depending on the region where these decompositions occur:

      (1) daughter-ion mode (determination of the decomposition ions of a given parent-ion): B/E = constant, MIKES (Mass-analyzed Ion Kinetic Energy Spectroscopy);

      (2) parent-ion mode (determination of all ions which by decomposition give an ion with a specific m/z ratio): 2 /E = constant;

      (3) neutral-loss mode (detection of all the ions that lose the same fragment):

QUADRUPOLES The analyzer consists of four parallel metal rods, which are cylindrical or hyperbolic in crosssection. They are arranged symmetrically with respect to the trajectory of the ions; the pairs diagonally opposed about the axis of symmetry of rods are connected electrically. The potentials to the two pairs of rods are opposed. They are the resultant of a constant component and an alternating component. The ions produced at the ion source are transmitted and separated by varying the voltages applied to the rods so that the ratio of continuous voltage to alternating voltage remains constant. The quadrupoles usually have a mass range of 1 a.m.u. to 2000 a.m.u., but some may range up to 4000 a.m.u. Although they have a lower resolving power than magnetic sector analyzers, they nevertheless allow the monoisotopic profile of single charged ions to be obtained for the entire mass range. It is possible to obtain spectra using three quadrupoles arranged in series, Q1, Q2, Q3 (Qserves as a collision cell and is not really an analyzer; the most commonly used collision gas is argon). The most common types of scans are the following:

      (1) daughter-ion: Q1 selects an m/z ion whose fragments obtained by collision in Q2 are analyzed by Q3;

      (2) parent-ion mode: Q3 filters only a specific m/z ratio, while Q1 scans a given mass range. Only the ions decomposing to give the ion selected by Q3 are detected;

      (3) neutral loss mode: Q1 and Q3 scan a certain mass range but at an offset corresponding to the loss of a fragment characteristic of a product or family of compounds.

      It is also possible to obtain spectra by combining quadrupole analyzers with magnetic or electrostatic sector instruments; such instruments are called hybrid mass spectrometers.

ION-TRAP ANALYZER The principle is the same as for a quadrupole, this time with the electric fields in three dimensions. This type of analyzers allows production spectra over several generations (MSn ) to be obtained.

ION-CYCLOTRON RESONANCE ANALYZERS Ions produced in a cell and subjected to a uniform, intense magnetic field move in circular orbits at frequencies which can be directly correlated to their m/z ratio by applying a Fourier transform algorithm. This phenomenon is called ion-cyclotron resonance. Analyzers of this type consist of superconducting magnets and are capable of very high resolving power (up to 1,000,000 and more) as well as MSn spectra. However, very low pressures are required (of the order of 10–7 Pa).

TIME-OF-FLIGHT ANALYZERS The ions produced at the ion source are accelerated at voltage V of 10 to 20 kV. They pass through the analyzer, consisting of a fieldfree tube, 25 cm to 1.5 m long, generally called a flight tube. The time (t) for an ion to travel to the detector is proportional to the square root of the m/z ratio. Theoret ically, the mass range of such an analyzer is infinite. In practice, it is limited by the ionization or desorption method. Time-of-flight analyzers are mainly used for high molecular mass compounds (up to several hundred thousand daltons). This technique is very sensitive (a few picomoles of product are sufficient). The accuracy of the measurements and the resolving power of such instruments may be improved considerably by using an electrostatic mirror (reflectron).

Signal Acquisition

      There are essentially three possible modes.

COMPLETE SPECTRUM MODE The entire signal obtained over a chosen mass range is recorded. The spectrum represents the relative intensity of the different ionic species present as a function of m/z. The results are essentially qualitative. The use of spectral reference libraries for more rapid identification is possible.

FRAGMENTOMETRIC MODE (SELECTED-ION MONITORING) The acquired signal is limited to one (single-ion monitoring, SIM) or several (multiple-ion monitoring, MIM) ions characteristic of the substance to be analyzed. The limit of detection can be considerably reduced in this mode. Quantitative or semi-quantitative tests can be carried out using external or internal standards (for example, deuterated standards). Such tests cannot be carried out with time-Rf-flight analyzers.

FRAGMENTOMETRIC DOUBLE MASS SPECTROMETRY MODE (MULTIPLE REACTION MONITORING, MRM) The unimolecular or bi-molecular decomposition of a chosen precursor ion characteristic of the substance to be analyzed is followed specifically. The selectivity and the highly specific nature of this mode of acquisition provide excellent sensitivity levels and make it the most appropriate for quantitative studies using suitable internal standards (for example, deuterated standards).

This type of analysis can be performed only on an apparatus fitted with three quadrupoles in series, iontrap analyzers or cyclotron-resonance analyzers.

Calibration

      Calibration allows the corresponding m/z value to be attributed to the detected signal. As a general rule, this is done using a reference substance. This calibration may be external (acquisition file separate from the analysis) or internal (the reference substance(s) are mixed with the substance to be examined and appear on the same acquisition file). The number of ions or points required for reliable calibration depends on the type of analyzers and on the desired accuracy of the measurement, for example, in the case of a magnetic analyzer where the m/z ratio varies exponentially with the value of the magnetic field, there should be as many points as possible.

Signal Detection and Data Processing

      Ions separated by an analyzer are converted into electric signals by a detection system such as a photomultiplier or an electron multiplier. These signals are amplified before being re-converted into digital signals for data processing, allowing various functions such as calibration, reconstruction of spectra, automatic quantification, archiving, creation or use of libraries of mass spectra. The various physical parameters required for the functioning of the apparatus as a whole are controlled by the computer.

APPENDICES • 2.7 X-RAY FLUORESCENCE SPECTROMETRY
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หมายเหตุ / Note : TP II 2011 PAGE 382-385