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Electron microprobe analysis (EMPA) or Electron probe microanalysis is a non destructive analytical technique that is used to determine the chemical composition of small selected areas of solid sample at micrometer scale.1 In 1950 R. Castaing developed Electron microprobe as his Ph. D dissertation.13 The Electron microprobe can be used for qualitative as well as quantitative chemical analysis. When a beam of electrons is focused on a solid sample surface, the materials present in the sample interact with the beam electrons to produce X-rays. The X-rays are detected by Energy dispersive spectrometer or Wavelength dispersive spectrometer and analysed to obtain quantitative chemical analysis of the sample.2

When a polished solid sample surface is bombarded by a focused beam of accelerated electrons the energy present in the incident beam liberates both energy and matter when it hits the sample. The energy released is in the form of heat, light, derivative electrons and X-rays. The electrons are mainly backscattered electrons and secondary electrons. These electrons are useful for surface imaging and obtaining an average composition of the specimen. The X- rays are formed due to inelastic collisions between the inner shell electrons in the sample and the incident beam electrons. As a result of the inelastic collisions the inner shell electron gets excited and it is ejected from its orbit leaving a vacancy. This vacancy is filled by electrons in the higher shell by shedding some energy in the form of X-ray. Except Helium and Hydrogen all elements in the periodic table emits specific X-rays when it is bombarded by electron beam.2These X- rays can be measured in Electron microprobe by two detectors-WDS which measures the wavelengths of the X-rays and the EDS which measures the energies of the X-ray. These measurements are used to find the chemical composition as well as the concentration of elements present in the sample.3, 4

Instrumentation:

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Schematic cut-away diagram of a typical microprobe.

Fig. 1 Schematic cut-away diagram of a typical Electron microprobe.14

The Electron microprobe has the following important components:

A) Electron-optical system or EOS: It consists of an electron gun and magnetic lens. An electron gun is the source of electrons which consists of a heated hairpin tungsten wire or lanthanum hexaboride which serves as a cathode and an accelerating anode to produce the electron beam. The electron beam passes through two electromagnetic lens and apertures to form an electron beam of 0.1-1 µm diameter. This electron beam is focused on the specimen. The lens controls the amount of electric current that passes through the Electron optical system. 4

B) Optical microscopes: The optical microscope as shown in the figure is used to locate the area on the specimen to be bombarded. During operation, with the optical microscope the electron beam position, its size and shape as well as small areas of specimen can be simultaneously examined.1

C) Sample stage: For proper scanning of the surface of the specimen, the sample stage has mechanism to rotate the sample as well as move it in mutually perpendicular direction.2, 5

D) Electron Detectors

Secondary electron detectors: The secondary electrons are detected by a scintillator -photomultiplier or "Everhart- Thornley" detector and it helps to acquire images similar to those obtained by Scanning Electron microscope and gives topological information.11

Back scattered electron detector: Solid-state diode detector is used to detect back scattered electrons and it helps to acquire images which show compositional contrast.2

E) X-ray spectrometer

I) Energy-dispersive spectrometer (EDS). Si (Li) detector and multichannel analyzer is used in EDS. To prevent permanent damage to the detector it is operated near liquid Nitrogen temperature. All the X-ray energies are measured simultaneously by energy dispersive spectrometer. 4

II) Wavelength-dispersive spectrometer (WDS)

WDS-RowlandCircle

Figure 2. Configuration of sample, analytical crystal and detector on the Rowland circle within the WD spectrometer.15

As shown in figure 2 above, the wavelength dispersive spectrometer consists of a diffraction crystal and X-ray detector known as proportional counter. When the diffraction crystal is hit by X-ray source, the X-rays are dispersed by Bragg reflection and measured by the detector. In electron microprobe in order to analyze a wide range of wavelength of X-rays several crystals with different d-spaces are used. Lithium fluoride (LIF), pentaerythritol (PET) and thallium acid phthalate (TAP) are the common crystals that are used that can measure wavelengths generated by elements in the periodic table from 11Na to 92U.Soap film can be used when wider d-spacing is needed for lighter elements. 3The X-rays are counted at a specific wavelength in WDS. The X-rays emitted by different elements in the sample are separated by their wavelength and then by using Bragg diffraction are diffracted at the detector.

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If we compare the EDS and WDS, the EDS is quick, versatile, inexpensive and very easy to use. It displays all the elements present in the sample within few minutes. However it has got poor resolution of spectra (130-155 eV)and low signal to noise ratio .As a result the spectrum obtained by EDS will have lot of interferences and the detection limit is 1000-3000ppm .Sensor in EDS cannot detect elements with atomic number less than 5. 3 The WDS on the other hand has very good spectral resolution (8eV) and so does not have spectral interference. Moreover it has high signal to noise ratio which makes it very sensitive to detection of trace elements. It has a low detection limit (10-100ppm).But it is expensive and has slow speed because the detector only analyzes one element at a time. In WDS the elemental composition of sample should be known before hand, because the elements have to be tuned at a specific wavelength.4

F) Vacuum system

In order to remove air from the electron column and achieve vaccum a pumping system consisting of a combination of mechanical and diffusion pumps are used in Electron microprobe. A pressure of 4x10-5 Pa (which is 3x10 -7 torr) provides excellent vaccum for electron microprobe chamber. Gun vaccum is essential to increase life of the tungsten filament and avoid arcing between the tungsten filament and anode. Also vaccum helps in better sample and electron interactions avoiding collisions between stray molecules and electrons of the beam help to form mean path length of electrons to be greater than the electron column length and prevent absorption of X-rays produced by the sample by air molecules.2

In EMPA, the output is in the form of X-ray spectrum. Nowadays with image analysis software spatial distribution of elements can be qualitatively and quantitatively mapped on sample surface. The energy spectrum of X-rays obtained by electron microprobe is made of X-ray lines characteristic of the elements present in the sample. By identifying the lines from their wavelengths and applying Bragg and Moselays law qualitative chemical analysis of the sample can be done.3

Braggs law: It shows a diffraction relationship between the wavelength of the incident X-rays, angle of incidence and d-spacing of diffracting crystal .5

It is expressed as n λ = 2d sinθ where

n = the order of reflection, λ = wavelength of incident ray, d = interplanar distance of the crystal and θ = angle of incidence and reflection of incident ray

Moselays law: The regular relationship between the atomic number of a material and its characteristic X-ray wavelength is given by Moseley's law and expressed as

l =B/ (Z-C) 2, where B and C are constants for each family of X-rays5.

Quantitative analysis is done by measuring the intensity of the X-ray lines generated by the sample .These intensities are compared to the intensities emitted by a standard reference material whose composition is known. Both the sample and the standards are analyzed under similar operating conditions. The computer is used to apply matrix corrections called ZAF corrections [atomic number (Z), absorption (A) and fluorescence (F).]The results are obtained as atomic proportions or Weight%.4 ,16

Thus the concentration of element is known by applying the formula

CA(sa) = [IA(sa)/IA(st)]CA(st)

Where ,CA(sa) = concentration in sample

CA(st) = concentration in standard

IA(sa) = X-ray intensity in sample

IA(st) = X-ray intensity in standard

IA(sa)/ IA(st) is known as the K ratio.The k ratio gives approximate weight fraction of the unknown.3

Applicability and Limitations

Sample preparation: Almost any solid material can be analyzed by Electron microprobe provided it is vaccum compatible. So wet and sticky samples cannot be analyzed. The sample surface needs to be flat and polished. Analysis of Grains or chips is done by mounting it on epoxy disk. The cross section of the material is exposed by polishing the disk halfway. Polishing removes surface imperfections that may interfere with interactions between sample and electron beam. The main goal of polishing is to obtain a flat smooth surface. Samples are then coated with a thin film of conducting material like carbon, gold or aluminum .17, 2

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Calibration of electron microprobe is done by using standard reference material of known compositions. Usually alloys, minerals or glass are used. Example Silicon and calcium are calibrated with Wollastonite (mineral).The standards are analyzed with the same instrument and under same instrumental conditions as the samples. The counts obtained are corrected for inelastic electron scattering and energy loss due to atomic number (Z), X-ray absorbance by the sample (A) and Fluorescence X-ray correction (F) by computer software. A correct analysis is usually obtained post calibration.16

Performance characteristics2: The detection limit and sensitivity of EMPA for light elements like C, F, N, and O is around 500-1000ppm and for heavier elements Na-U is about 300ppm.But again this is dependent on X-ray counting time and mean atomic number of the matrix. The spatial resolution for quantitative analysis is of the order of 1-3 microns. Back scattered and secondary electrons have a resolution of approximately 100-200nm depending on the beam current and accelerating voltage. The accuracy depends on the similarity of the sample and the standards. The accuracy up to ±1% (relative) can be obtained for major elements. Trace elements and light elements have less accuracy. Moreover all elements except H, He, and Li measurable .EMPA has a sensitivity of ±0.2 at.% Also the method is fast, whole analysis takes 1-2 min. and has high precision (<1% relative error)

The Electron microprobe(EM) uses an electron beam to excite (ionize) the electrons in the atoms of the sample in the same way as Scanning electron microscope(SEM) .But, Electron Microprobe does not detect the electrons released due to ionization but measures the actual energy lost in the ionization process. The electron microprobe also has advantage of quantitative chemical analysis which is not possible in SEM.

Advantages: One of the characteristic features of electron microprobe that distinguishes it from electron microscope is that it is equipped with crystal spectrometers that helps to determine quantitative chemical composition(WDS) at high sensitivity.3 Moreover chemical analysis with spatial distribution of very small species( as small as 1-2 micron diameter) is possible with electron microprobe. This helps the user to analyze extremely small single phases in samples (e.g. minerals in rock) in situ. EMPA is a spot analytical technique in which the chemical information is collected only from a small volume of the entire sample. The small volume is representative of the entire sample, provided the sample is homogenous. In the electron microprobe analysis, though the sample is bombarded with electron beam no change in volume or destruction of the sample occurs. As a result the same sample can be analyzed again. The various detectors in this instrument are useful for imaging surface as well as internal compositional structures.

Limitations: The quantitative measurements obtained in EMPA are Quantitative measurements depends on standards. Complex organic molecules and minerals with same composition but different crystal structures are difficult to analyze. Vaccum incompatible elements and the lightest elements (H, He and Li) cannot be analyzed. Elements generating X-rays with overlapping peak positions (by both energy and wavelength) require separation. Microprobe analysis is reported as oxides of elements and so proportions of cations and mineral formulae require recalculation by stoichiometric rules.

Applications: Information about physical and chemical nature of surfaces is provided by Electron Microprobe. EMPA with its ability to obtain qualitative and quantitative about surfaces has many applications to studying phase in metallurgy and ceramics. With EMPA grain boundaries in alloys can be determined, diffusion rate of impurities in semi conductors and occluded species in crystals can be measured, and active site on heterogenous catalyst can also be determined5

Research applications of EMPA:

A)Application of electron microprobe in analysis of ash samples produced as a byproduct of gasification.[Tivo B. Hlatshwayo, Ratale H. Matjie, Zhongsheng Li, Colin R. W(2009).Mineralogical Characterization of Sasol Feed Coals and Corresponding Gasification Ash ConstituentsEnergy & Fuels 23 (6), 2867-2873)]8

This research study was based on Sasol Lurgi fixed bed ,dry bottom gasifier which is used to convert coal into combustible gas.During the gasification process ,under high temperature and pressure conditions the minerals present in the coal interact with any noncoal particles forming ash as a byproduct.The ash is a mixture of fine and coarse particles. The physical properties of the ash -hardness, abrasiveness, particle size depends on the minerals present in the coal that is feeded in the gasifier and the operating conditions. The researchers in this study evaluated the mineralogical, physical and chemical properties of coal and ash particles produced during gasification at different temperatures by the gasifier tests. X-ray diffraction analysis was used to find the mineral percentage present in the ash, clinker (incombustible residue left after the combustion of coal) and unaltered non coal particles like rocks. X-ray fluorescence analysis was done to know chemical composition of ash and stone samples. EMPA showed the mineral composition of ashes expressed as weight % of different elements .EMPA analysis was done on samples of ash, heated stones and clinker samples .The samples were taken ,mounted into polished sections and coated with carbon .Back scattered electron images were also collected which were representative of each sample and provided textural observations..The images showed the mode of occurrence of the important minerals in ash samples.

The components of the ash can have effect on the wear of the coal or ash handling systems. The EMPA analysis of the ash helped the researchers to know the source of abrasion or predict the abrasive characteristic of ashes based on mineral composition. This data would have implications in finding the wear rates of susceptible gasifier components and development of maintenance methods to prevent such wear .Thus

B) EMPA used to study effect of precursors on the activity of catalyst

[Qian, L., Yang, J., Zhi-Dong, J., & Wen-De, X. (2007). Effects of Precursors on Preparation of Pd/-alumina Catalyst for Synthesis of Dimethyl Oxalate. Industrial & Engineering Chemistry Research, 46(24), 7950-7954]

Synthesis of Dimethyl oxalate is done by CO coupling reaction in which Palladium catalyst supported on alpha alumina is very effective.The Palladium catalyst that are prepared are usually made using palladium chloride as a precursor and the best loading of palladium is confirmed to be 1.0 wt%.The distribution of active components in catalyst greatly influences the performance of catalytic reactions.In this study the researchers wanted to investigate the the effect of two palladium precursor-palladium chloride and palladium nitrate on preparation of Pd/alpha alumina catalyst and differences in preparation steps required when using different precursor.At the same time they wanted to determine the effect of precursor on distribution of palladium on alpha alumina supported catalyst and the loading amount of palladium on the pellet.

They prepared two different catalysts using palladium chloride and palladium nitrate as precursor under different temperature and pressure conditions and by loading different amount of pd.Inductively coupled plasma was used to know the loading amount in the various prepared catalyst and Thermogravimetric analysis was done to know the thermal decomposition temperature of the catalyst precursor. The dispersion and reduction of palladium was measured by Xray photoelectron microscopy(XPS) and CO chemisorption respectively.In order to measure the distribution of Palladium in series of catalyst (with palladium chloride and palladium nitrate as precursor )within the alpha alumina support pellet scanning Electron Microprobe was used.The pellets were mounted in plastic,sectioned equatorially and the cross sections were scanned by electron microprobe.The pictures taken by electron microprobe showed the'egg shell' distribution of Pd in palladium nitrate catalyst and tree ring type distribution in Palladium chloride catalyst.The concentration of palladium in the the egg shell as well as tree ring was determined as weight% by EMP.

The researchers did a good job in finding the benefits of palladium nitrate over palladium chloride as precursor-reduced temperature for calcination and reduction avoiding sintering of Pd particles,avoiding release of HCl ,and optimum egg shell distribution of palladium in support pellet which reduces loading of palladium form 1% to 0.1 weight %.

Electron microprobe proved to be a valuable tool in obtaining the images of palladium pellets and also determining the concentration of Palladium in these pellets .

C)Use of electron microprobe in paleontology to study elemental abundance in fossils.[Boyce, C., Hazen, R., & Knoll, A. (2001). Nondestructive, in situ, cellular-scale mapping of elemental abundance including organic carbon. Proceedings of the National Academy of Sciences of the United States of America, 98(11), 5970.]7

Biological remains are preserved in premineralized fossil. Chemical analysis of the organic matter in premineralized fossils gives valuable insight for tissue and cell level study. Elemental abundances can be mapped by an Electron Microprobe at micrometer scale. It is difficult to obtain high resolution maps of light elements by EMPA. Light elements have relatively long wavelengths of characteristic X-rays, less yield of X-rays, and high absorption of X-rays in the sample. 2

The organic matter in these fossils also contains carbon. Carbon being a light element is difficult to analyze by EMPA along with problems posed when differentiating between sample carbon and coating of carbon on sample. So the researchers in this study modified the standard electron microprobe procedure .Rather than usual 10-30nV electron beam current they increased the current to approximately 300nV in order to increase the sensitivity for light element detection. Secondly rather than coating the fossil sample with carbon they coated the sample with Aluminium which is a cheap, stable, safe light metal that allows good conductance without high absorption of electrons. The aluminium coat eliminates the difficulty encountered in discriminating between carbon in coating and native carbon in the sample. The samples used in this study were in general vascular plants stems -Proterozoic cyanobacteria (geological eon )to Neoangiosperms which were preserved by early diagenetic calcifications and showed good details at cellular level when viewed by optical microscope. The silicified material examined in this study belonged to Draken formation, Rhynie chert, Olentangy Shale, Serian volcanic formation, Princeton chert, Vantage sandstone and lost Chicken creek chert (Chert - sedimentary rock) The fossil samples were of standard rectangular 1 inch by 2 inch or circular 1 inch polished thin and thick sections which were coated with aluminium by vaporizing aluminium foil placed on tungsten wire of vaccum coating device. Electron microprobe was then used to obtain semi quantitative analysis and compositional maps of elemental abundances in the fossil sample.

The probe measurements in the results show a very high content of silica of approximately 70% in preserved cell walls which is very high. This may be due to averaging over 1-3 micrometer width of electron beam and background x-ray fluorescence from surrounding matrix. The researchers did a good job in finding the distribution of major and trace elements in the matrix of the fossil and organic carbon present in the fossil. This allowed them to study the decay and formation of silicified fossil (taphonomic process) and the role of trace elements in starting silica precipitation and preventing organic degradation. Also by doing EMPA on fossil sample researchers come to know beforehand if sufficient organic material is present in the sample and is actually localized in tissue .This information from mapping of fossil is valuable before subjecting the sample to destructive bulk analytical techniques like isotope ratio mass spectrometry, NMR, pyrolisis based organic analysis etc.

The researchers did a good job in modifying the procedure to obtain fine scale mapping of trace elements like carbon and in present day aluminium is used to coat the samples for EMPA.