Contents

1
Fundamentals of X-Ray Fluorescence Analysis
Virgilio F. Nascimento Filho 5

2
Sample Preparation
Graciela Custo, Dora V. de Leyt and Cristina Vázquez

3
Chemometrics in Atomic Spectroscopy
Alejandro C. Olivieri

4
Total Reflection X-ray Fluorescence Analysis - Instrumentation
Peter Wobrauschek

5
Total Reflection X-Ray Fluorescence Analysis of Low Z elements:
Developments and Applications
Christina Streli

6
Direct Analysis of Biological Samples by Total Reflection X-Ray
Fluorescence
Lué-Merú Marcó P.

7
X-Ray Spectrometry for Analysis of Atmospheric Particulate Matter:
Detection Limits Versus Legal Levels
R. Van Grieken, Y. Makarovska, K. Van Meel and A. Worobiec
8
In-Situ XRF Analysis
Andrzej Markowicz

9
Particle Induced X-Ray Emission (PIXE)
Javier Miranda

10
Polymeric Materials by TXRF
Susana Boeykens
 1
Fundamentals of X-Ray
Fluorescence Analysis

Virgilio F. Nascimento Filho

Abstract
X-ray fluorescence is an analytical technique that has been used for both, qualitative and quantitative
evaluation of chemical composition in various types of samples, coming from different fields such as
agriculture, industry, geological, environmental, etc. Being a non destructive and instrumental, this
technique allows the analysis of several elements simultaneously, in a fast way and at a low cost. It
has a potential scope of application in many fields where the correlation between toxic and essential
elements is needed.
The purpose of this writing is the introduction to X-ray fluorescence (XRF) and its difference modes:
energy dispersive X-ray fluorescence (EDXRF), wavelength dispersive X-ray fluorescence (WDXRF).

1.- Introduction
The multi-elemental and instrumental analysis by X-ray fluorescence (XRF) is based on the measure
of characteristic X ray intensities emitted by chemical elements, which are sample constituent, when
are adequately excited. Up to 1966, the XRF was only made by wavelength dispersive spectrometers
based on Bragg Law, which need an accurate and synchronized movement between a diffraction
crystal and a detector [60]
The development of high resolution semiconductor Si (Li) detectors, which are capable to
discriminate X-rays of close energy, made possible the coming up of energy dispersive X-ray
fluorescence (EDXRF), also known as non dispersive, with less expensive instrumentation and more
practical use. [14, 57, 63,132]
The XRF technique is being used mainly on solid samples, allowing the sequential or simultaneous

3
determination of the concentration of various elements, with no need of sample destruction generally,
that is, as instrumental way, without any previous chemical treatment. In case of liquid samples, it is
possible to employ a pre-concentration procedure by using ionic exchange, precipitation, chelate
formation, etc.
This technique shows a high analytical speed for semi-quantitative analysis on samples of agriculture,
geological and environmental interest when X-ray tubes are used for excitation. X and/or gamma rays
radioactive source emitters of low energy (55Fe, 57Co, 109Cd, 238Pu, 241Am) can also be used, replacing
excitation by X-ray tubes making the analysis much simpler, cheaper and much easier operation,
with the disadvantage of analytical sensitivity loss. Likewise, the EDXRF by radioactive sources
excitation has found uncountable applications, mainly in the scope of industry, geology and mineral
prospecting, where high analytical sensitivity is no necessary.
An X-ray fluorescence variant of energy dispersive, named Total Reflection (TXRF), developed in
the last few years is successfully applied in the following fields: trace element analysis (nanograms or
part per billon range) in liquid samples, investigations linked with environmental control,
oceanography, biology, medicine, industry, mineralogy, etc, and specifically, on superficial waters
(rain, river and marine) and groundwater, biological fluids, forensic field and for the quality control
on high-purity products.
The TXRF technique can also be applied on solid materials (soil, sediment, air filter, particulate
material, etc.), but it must be preceded by a chemical digestion and appropriated dilution, as those
used for flame photometry, spectrophotometry, atomic absorption/emission (AES) and their variant
(ICP/AES, ICP/MS) in this type of samples. TXRF shows the great advantage that needs very small
amounts (milligram order) for the digestion [71].
Due to the interactions among the elements in the sample [134, 138, 158, 173], quantitative analysis by
EDXRF, as well as WDXRF, has the disadvantage of requiring matrix effects correction (absorption
and enhancement) of the characteristic X-rays.
These effects do not happen in the TXRF, because are used very small amounts of samples (less than
10 µL of liquid samples and evaporated or smaller than 10 mg of solid samples) deposited on a
support, as thin films. Therefore for quantitative analysis a simple lineal regression between the
intensity of X-rays and the concentration of the analyte can be used.
EDXRF, instrumentally cheaper than WDXRF, become more used technique in the last years, as
much for the qualitative as quantitative analysis. Detection limits from 1 to 20 µg/g for solid samples,
without chemical treatment and on the order of 1 to 20 µg/kg for liquid samples with pre-
concentration treatment, can be obtained. Due to the simplicity, speed and analytical cost, EDXR
gives great interest for the analysis of biological samples, because of the metabolic interactions
existing between the macro and micro-elements.
Among the advantages of the X-ray fluorescence for the elemental chemical analysis it is possible to
be mentioned: (a) multi-elemental fast analysis, very important due to the interdependence between
micronutrients in the biological systems, (b) simplified preparation of the sample, (c) detection limits
within the ones demanded in the analysis of many biological samples and (d) possibility of doing
automatization.

2.- Principles of the X-Ray Fluorescence

The analysis by X-ray fluorescence is a method for instrumental cuali-quantitative analysis, based on
the measurement of the characteristic X-ray intensities (number of X-ray photons detected per unit
area per unit time) emitted by the elements that constitute the sample [19, 57].
X-ray emitted by X-ray tubes or sources that emit X or Gamma radiation, excite the elements present
in the sample. The emitted spectral lines are characteristic of the corresponding element and the
intensities are related to the concentration of the element in the sample.

4
When an X-rays beam impinges on a certain material causes the excitation of the constituent
elements, as a result of this, electron from the atoms are expelled from the inner levels, and the
electrons from levels farther out fall into vacancies. Each electronic transition constitutes an energy
loss which appears as a well defined characteristic energy and for each element. Summarizing: the
analysis by X-ray fluorescence consists of three phases: excitation of the elements that constitute the
sample, dispersion characteristic of X-ray emitted by the sample and detection of those X-ray.

2.1.- Excitation of the Elements

In order to cause the emission of characteristic X-rays of the elements that constitute the sample, the
excitation can be made in several ways: excitation by accelerated particles such as electrons, protons
or ions; by radioisotope sources which emit X rays, α, β or γ radiation; and X-ray tubes. In these
processes, the instrumentation needs electronics devices to produce extremely stable high tension, and
therefore they are sophisticated and expensive. When radiation sources are employed, instrumentation
is cheaper and compact, but requires radiological shielding to retain radiation.
The most effective radioisotope sources used in XRF are those that the disintegrated process is by
electronic capture, such as 55Fe (half-life of 2.7 years; X-ray Mn-Kα emitter of 5.9 keV) and 109Cd
(half-life 1.27 years; X-ray Ag-Kα emitter of 22.2 keV); or by alpha emission, like 238Pu (half-life
86.4 years, X-ray U-L emitter of 13.6 and 17.2 keV) and 241Am (half-life 428 years; X-ray emitter
Np-L of 13.9 and 17.7 keV, and gamma radiation of 59.5 keV). Table 1 shows the radioisotopes used
as sources of excitation in EDXRF.

Table 1. Main radioisotopes used as sources of excitation in EDXRF [57]

Half-Life Photon Energy Emission
Radioisotope Disintegration Process*
(years) (keV) (%)
55
Fe 2.7 EC 6 (Mn K) 28.5
238
Pu 86.4 α 12-17 (U L) 13.0
109
Cd 1.29 EC 22 (Ag K) 107.0
88 γ 4.0
125
I 0.16 EC 27 (Te K) 138.0
35 γ 7.0
210
Pb 22 β 11-13 (Bi L) 24.0
47 γ 4.0
241
Am 428 α 14-21 (Np L) 37.0
60 γ 36.0
153
Gd 0.65 EC 41 (Eu K) 110.0
70 γ 2.6
97 γ 30.0
103 γ 20.0
57
Co 0.74 EC 6,4 (Fe K)
14 γ 8.2
122 γ 88.9

5
 136 γ 8.8
EC= Electronic capture α = alpha particle
β = beta particle γ = gamma ray

In order to produce characteristic X-ray, it is necessary to irradiate the sample with enough energy to
eject electrons from the inner shell of the atom, for example K shell. The energy must to be higher
than the binding energy of the electron.
This energy can be calculated using the Bohr theory for the hydrogen and Moseley law. Equation 1 is
used to calculate approximately the energy for electron from K and L shells.

m.e4 (Z − b )
2
E= Eq(1)
8.ε 0 .h 2 .n 2
2

where:
E = energy of electronic bound (joules),
m = mass of the electron = 9.11 10-31 kg,
e = electronic charge of the electron 1.6 10-19 coulombs,
Z = atomic number of the emitting element,
b = Moseley´s constant, 1 and 7.4 for shells K and L respectively
εo = electrical permittivity in the vacuum = 8.8534 10-12 coulombs.newton-l.m-2,
h = Planck´s constant (6.625, 10-34 joules s),
n = principal quantum numbers (for K, L, M are n = 1, 2, 3, respectively).

Replacing in Eq(1) the correspondent constant values according to the International System of Units,
the energy of X-rays (in joules) is given by:

E = 2.18 *10−18
(Z − b )2 Eq(2)
n2

and replacing 1 eV = 1.6 10-19 joules, Eq(2) can be rewritten in terms of electronvolts as follows:

E = 13.65
(Z − b)2 Eq(3)
n2

Eq(3) shows that the binding energy in one shell is directly proportional to the square of atomic
number Z of the element. For instance, the corresponding energy to eject electrons from the K shell of
Al, Fe and Te (atomic numbers 13, 26 and 52, respectively), are 1.560, 7.114 and 31.814 keV. For L
shell will be 0.074, 0.723 and 4.612 keV, respectively.

6
After ionization, the place of the missing electron is filled by an electron from the neighboring outer
shell, and an X-ray of a characteristic energy is emitted, with an energy value depending on the
difference between the two shells. Consequently, the energy of X-ray also is directly proportional to
the square of atomic number Z of the excited element, when same quantum jump is considered.

E x = E ni − E nf Eq(4)

where:
Ex = energy of emitted characteristic X-ray, and
Eni and Enf = energies of the electron in the levels initial and final, respectively.

In EDXRF modality, this equation is fundamental in order to understand the proportionality between
the energy (or the amplitude of the produced electronic pulse in the detector) and the analyzed
element).
In WDXRF systems according to Bragg law for diffraction crystals, is more interesting to have the
relation between the wavelength of characteristic X-ray and the atomic number of the analyzed
element. In this case, in Eq(1) the X-ray energy can be replaced by the corresponding wavelength λ,
using Planck´s equation:

h.c
E= Eq(5)
λ
where:
c = velocity of the light in vacuum = 3 108 m s-l,
λ = wavelength (m).

Replacing the constant h and c by its values, Planck´s equation can be rewritten in the following way:

Eλ = 1240 eV nm Eq(6)

From Eq(3) and Eq(5) it is evident that the wavelength λ of emitted X-ray is inversely proportional to
the square of atomic number Z of the excited element.
As example, applying Eq(2) or Eq(3) is possible to calculate the emitted energies for molybdenum X-
rays (Z = 42): knocking out an electron of K level, an electron of L level can fill this vacancy, the
transition is from n = 2 to n = 1 (Kα line). In this case, from Eq(3) an energy value of 17.78 keV is
obtained (from tables the corresponding value is 17.42 keV [46]). Using Eq(5), it is possible to
calculate the corresponding wavelength for the Mo Kα line which is 0.0723 nm (the experimental
value by Moseley relation is 0.0721 nm).
For iron (Z = 26) and considering the quantum jump from n = 2 to n = 1, the energy of X-rays is 6.40
keV or 0.194 nm (Kα line). If the quantum jump is from M level to K level, the corresponding
electronic transition produces Kβ X-ray of 7.06 keV or 1.76 nm, as show in Figure 1.

7
 Energy (keV)

Atomic number

Figure 1. Relationship between absorption edge for K, L and M shells and atomic number

In fact, those considerations are somewhat simple, because it was considered that the electrons of
same shell have same energy; but it is known that the electrons are distributed in sub-levels of close
energies; this can be visualized in Figures 2 and 3 for iron and molybdenum, respectively. For iron,
an electron transition LII-K produces a characteristic Kα2 X-ray of 6.391 keV, and for electron
transition LIII-K the energy of Kα1 X-ray is 6.404 keV; transition LI-K does not happen because is a
"prohibited transition".

IRON 26Fe

Figure 2. Energetic levels and relative emission intensities for iron

8
 MOLIBDENUM 42Mo

Figure 3. Energetic levels and relative emission intensities for molybdenum

Transitions LIII-K and LII-K have very close energies and it is impossible to separate even with high
resolution detector, such as a Si(Li). For this reason both transitions are included in a single line, with
average energy of 6.40 keV. Same happens for energies coming from quantum jumps M level to K
level, producing a single line that can be seen in Figures 2 and 3.
For heavy elements, the existence of other sub-levels make more complicated the spectra, as can be
seen in Figure 3 for Mo. For this reason, a special designation is needed. The most used since 1965 is
Siegbahn [132] notation as is shown in Figure 4. The symbol of an X-ray spectral line consists of: (a)
the chemical symbol of the element, (b) the symbol of the final level (K, L, M, N, etc.), (c) a Greek
letter with a numerical subscript. IUPAC [61] recommends the direct annotation (final level - initial
level), that is, to replace the Kα2, Kα1 and Kα notation for Siegbahn designation by K-L2, K-L3 y
K-L, respectively.

Siegbahn IUPAC Siegbahn IUPAC

Kα1 K-L3 Mα M5-N7
Kα2 K-L2 Mα M5-N6
Kβ1I K-M3 Mβ M4-N6
Kβ2II K-N3 Mγ M3-N5
Kβ2 K-N2 Mξ M4,5-N2,3
Kβ3 K-M2
Kβ4I K-N5
Kβ4II K-N4
Kβ4x K-N4

9
 Kβ5I K-M5
Kβ5II K-M4

Siegbahn IUPAC Siegbahn IUPAC

Lα1 L3-M5 Lγ1 L2-N4
Lα2 L3-M4 Lγ2 L1-N2
Lβ1 L2-M4 Lγ3 L1-N3
Lβ2 L3-N5 Lγ4 L1-O3
Lβ3 L2-M3 Lγ4’ L1-O2
Lβ4 L1-M2 Lγ5 L2-N1
Lβ5 L3-O4,5 Lγ6 L2-O4
Lβ6 L3-N1 Lγ8 L2-O1
Lβ7 L3- O1 Lγ8’ L2-N(7
Lβ7’ L3-N6,7 Lη L2-M1
Lβ9 L1-M5 Ll L3-M1
Lβ10 L1-M4 Ls L3-M3
Lβ15 L3-N4 Lt L3-M2
Lβ17 L2-M3 Lu L3- N6,7

Figure 4. Siegbahn and IUPAC notations for characteristic X rays

ENERGY (Kev)

ATOMIC NUMBER

Figure 5. Energy variation of Kα, Kβ, Lα and Lβ characteristic X rays with the atomic number

10
Figure 5 shows the variation of characteristics X rays Kα, Kß, Lα and Lß with the atomic number.
Sometimes, when the atom rearranges by filling the vacancy, the originated energy may be released
as an electron instead of an X-ray photon, this process is known as Auger effect (Figure 6). The
energies are also characteristics of the emitting element. Auger effect provides the basis of the Auger
spectrometry method. This effect is more common in low atomic number elements.

incident
radiation

Figure 6. Schematic representation of the Auger effect for magnesium

In this context, it is possible to define the fluorescence yield as the number of emitted photon by unit
time divided the number of vacancies formed in a given shell during the same period. Figure 7 shows
the variation of the fluorescence yield respect to the atomic number for K, L and M shells. It is clearly
noted that is lower for light elements (atomic number less than 20) for K shells, for L shell until
atomic number 60 and for M shell practically for all the elements.
FLUORESCENT YIELD (W)

ATOMIC NUMBER

Figure 7. Fluorescent yield for K, L and M shells respect to the atomic number

2.2.- Dispersion of X-Ray

The used methods of dispersion in most of the X-ray spectrometers can be classified in two
categories: wavelength dispersion (WDXRF) and energy dispersion (EDXRF), also known as
nondispersive, schematically illustrated in Figure 8.

11
 SAMPLE
X-RAY
TUBE

COLLIMATOR
CRYSTAL

WAVELENGTH DISPERSION WDXRF

SAMPLE

RADIOISOTOPE
SOURCE

ENERGY DISPERSION EDXRF

Figure 8. Schematic representation of wavelength dispersive and energy dispersive modalities

In wavelength dispersion method (WDXRF), the separation of the characteristic X-rays on the basis
of their wavelengths, is made by using diffraction crystals according to the Bragg law. In this case,
the selected wavelength can be calculated by the Bragg equation:

n.λ = 2.d .senθ Eq(7)

where:
λ = wavelength of the diffracted X-ray (nm),
d = interplanar spacing of the diffraction crystal (nm),
θ = the Bragg angle, is the angle between the incident X-rays and the diffracted planes (deg)
n = integral number 1, 2, 3, .., indicating the diffraction order

Crystals are, in general, available in more than one crystallographic orientation, called Miller indexes,
and are represented with three numbers (hkl). For example, in order to select X rays emitted by Fe
Kα (6.4 keV or 0.194 nm) by using a lithium fluoride crystal, LiF(220) d = 0.201 nm), the angle θ
between X-rays incidents and the surface of the crystal must be 28º 50'. A simplified design of the
characteristic X-rays diffraction of Fe Kα can be seen in Figure 9.

12
 BRAGG LAW
Nλ = 2D.sen θ → θ 0 arc sen (λ/2d)

LiF (200) CRISTAL d= 0,201 nm

Fe Kα 6,40 Kev λ = 0,194 nm
∴ DIFRACTION ANGLE θ = 28 50´ → 2θ = 57 40´
0 0

Crystal LiF (200)

X-RAY
INTENSITY

2θ ANGLE

Figure 9. Schematic representation of Bragg law for Fe, Mn and Co

It must be noted that incidence angle θ must be changed according to the diffracted element. For
example, to select X-rays of Co (0.179 keV or 6.92 nm), the angle must be changed to 26º 26', using
same crystal of LiF (200).
After the selection of desired X-rays, they must to be detected. For this reason the detector is placed
in the direction of the diffracted X-ray beam. In Figure 9 is possible to observe how the diffracted
beam forms an angle of 2θ in relation to the direction of the incident beam and also it is mandatory to
have a synchronism between the rotation of the crystal and the X-ray detector (Figure 10). In Figure
10 can be seen a graphical registry of the X-rays spectrum obtained in a WDXRF instrument for a
metallic sample.
The other modality in X ray spectrometry is energy dispersion (EDXRF). In this case, X-rays are
selected through the produced electronic pulses in an appropriate detector, the total charged collected
is linearly proportional to the X-rays photon energies. The more used detectors are the scintillation
counters of NaI(Tl) and the semiconductors of Si(Li), Ge(Li) and hyper-pure Ge.

13
 WDXRF SPECTROMETER

SAMPLE

X-RAY
TUBE

ROTATION
DIFRACTOR
CENTER
CRYSTAL

LiF (220) CRYSTAL

d = 0,142 nm
INTENSITY

Figure 10. Wavelength dispersive X ray system showing the synchronism between crystal and detector.
Wavelength dispersive X ray spectrum from a Cu/Ag alloy covered by a film of Ni and Cr[38]

Figure 11 is a scheme of an EDXRF spectrometer using a radiation source for the excitation of the
sample.

SAMPLE

Polarization source
+ amplifier
Multichanel analyzing card

Figure 11. Simplified scheme of an energy dispersive system with a radioisotope as source of excitation

14
Figure 12 shows a spectrum of pulses produced in a EDXRF spectrometer, using a radiation source of
55Fe for excitation and a semiconductor detector of Si(Li) for X-rays detection, in a sample of bovine
liver and fruit leaves, excited in different ways[37]. Figures 13 and 14 are displayed spectrums of clay
and sand soils, with and without acid treatment, excited with radiation sources of 55Fe y 109Cd [140, 141],
respectively.

Mo tube

Cd-109 Fe-55

Protons

Fe-55
Mo Tube

Figure 12. EDXRF spectra from bovine liver and orchard leaves using different excitation conditions as
is shown. Detection: Si(Li)

15
 INTENSITY

CHANNEL NUMBER

Figure 13. EDXF soil spectra with (a) and without (b) acid treatment. Excitation: 3,7 GBq 55Fe [140, 141],
detection: Si(Li)
INTENSITY

CHANNEL NUMBER

Figure 14. EDXRF from sand soils. Excitation: 370 MBq , 109Cd [140, 141]. Detection : Si(Li)

2.3.- Detection and Measurement of X-Rays

In WDXRF systems, proportional and scintillation crystal NaI(Tl) are normally used. The
proportional detector is used for low energy X-rays of (in the range of 1 to 15 keV), where it has a
high efficiency; scintillation crystal is used for high energy X-rays (100 to 15 keV).
It must to be remembered that in that system, the separation or selection of X-rays is made through
the diffraction crystal and a high resolution detector it is not needed. The detector must have a low
died time, for that reason normally a proportional detector or scintillation crystal are used discarding
Geiger-Mueller detector.
In EDXRF systems, a high resolution detector is mandatory in order to produce electronic pulses
proportional to the energies of X-rays. In this case, the most used is the silicon detector activated with

16
lithium, Si(Li), and some times the one of hyper-pure germanium, Ge. Figure 15 shows the resolution
for the detectors mentioned (except GM detector) for X-rays of 25.2 and 22.1 keV emitted by silver.

COUNT RATE

ENERGY (Kev)

Figure 15. Detector resolution according different X- ray detectors, for silver radiation

Si(Li) detector is the most used for Kα X-rays detection emitted by elements of atomic number in the
range of 13 to 50 (Sn) and L X-rays of the heavy elements. Due to its low efficiency for low energy
X-rays, they are not advisable for the detection of X-rays emitted by elements with atomic number
less than 13.
For high energy K X-rays emitted by elements of high atomic number (Z > 50), it is more advisable
the use of Ge(Li) detector due to its greater efficiency in relation to the detector of Si(Li) in this
region of energy. Figure 16 shows the efficiency of detection of semiconductor detectors based on the
X-rays energy. The main disadvantage of these detectors is the high mobility of lithium at room
temperature, causing the deterioration in the characteristics of the detectors, consequently the detector
must be maintained perpetually at liquid nitrogen temperature (- 180 oC).
In WDXRF analysis some electronic components, apart from the proportional detectors and
scintillation crystal, are used. Thus, the pulses produced by those detectors are sent to a counter,
hyphenated to a graphic recorder and, some times, to a printer. Graphic obtained with this system are
shown in Figure 10, where the height of the registered peak is proportional to the intensity of X-rays
emitted by an element and this, proportional to the concentration of the element in the sample.
In EDXRF analysis a multi-channel analyzer of pulses is used, also connected to a graphical recorder
and a printer. In that case, the area of the peaks is proportional to the intensity
of emitted X-rays by an element and consequently proportional to its concentration in the sample
(Figures 12 to 14).
WAVELENGHT (nm)
DETECTION EFICIENCY (%)

ENERGY (Kev)

Figure 16. Detector efficiencies for Si(Li) and Ge(Li), 3 and 5 mm thickness, for different Be windows,
according to X- ray energy

17
In both cases, the current tendency is to replace the graphical recorders by microcomputers, that
include software to capture the signals from the detectors and calculate peaks height and the peaks
area, and consequently calculate the concentration of the elements in the samples [ 60, 166, 172 ] . Finally
printed data of the samples could be obtained by mean of a printer.

3.- Fundamental Equation

Using mono-energetic excitation, a simple relation between the intensity of a characteristic line (Kα
and Lα) and the concentration of an element in the sample can be obtained. Assuming a homogenous
sample with uniform thickness D and rejecting the enhancement effects, the intensity of a Kα line of
an element in a layer dx at an x depth (Figure 17) is due to the three probabilities:
1. the probability P1 of the excitation radiation to reach the dx layer at x depth :

Figure 17. Schematic representation of monoenergetic excitation geometry

where:
µo = mass attenuation coefficient (cm2 g-1) at the energy of the incident radiation,
ρo = matrix density (g cm-3)
θo = incident angle (between the incident beam and the sample surface).
The value µo can be calculated from the sum of the product of the attenuation coefficient of each
element present in the sample and its weight fraction. Figure 18 shows the relationship between the
attenuation coefficient and the energy for Ca, Cu and U. The absorption discontinuities (absorption
edges) for the K level of Ca and Cu, L level (with 3 sublevels) and M level (5 sublevels) for U are
shown. WAVELENGTH (nm)

1. ABSORPTION
MASS ABSORPTION COEFFICIENT (cm2/g)

ENERGY (Kev)

Figure 18. Variation of mass absorption coefficient with the energy or Ca, Cu and U

18
2. The probability P2 of the excitation radiation for producing vacancies in the atoms of the
element of interest contained in the dx layer originating characteristics X-ray:

⎛ 1⎞
P2 = τ .⎜⎜1 − ⎟⎟.w.f .ρ .dx Eq (9)
⎝ j⎠

where:
τ = photoelectric mass absorption (cm2 g-1) for the element of interest at the excitation energy,
j = jump ratio K → L,
w = fluorescent yield for K shell,
f = fraction of photons emitted as Kα characteristics X-ray,
ρ = density (g cm-3) or concentration of the element of interest contained in the dx layer.

The photoelectric mass absorption coefficient at absorption edge value can broken down into a sum
of probabilities of ionizing each shell, but also can be divided in two values one upper, the probability
to ionizing K, L, M, etc. shells and the other one lower, the probability to ionizing all the shells
except the K shell.
In this way, it can be defined the ratio between the upper and the lower values known as jump ratio
(Figure 19), and it is the probability to ionizing all the shell related to the others L, M, etc. On the
other hand 1-1/j represents the probability to ionizing K shell related with K, L, M, etc shells; and
τ.(1-1/j) represents the number of ionizations in the K shell (Figure 19).

JUMP RATIO
JUMP RATIO

ATOMIC NUMBER

Figure 19. Jump ratio values for different atomic numbers

The fundamental parameters τ, j, w and f for a given element depend only of the excitation energy nd
can be included in the term K known as fundamental parameter constant. In this sense, Eq(9) it can be
written it as follows:

19
 P2 = K.ρ.dx Eq(10)
where

⎛ 1⎞
K = τ .⎜⎜1 − ⎟⎟.w.f Eq(11)
⎝ j⎠

3. The probability P3 of the characteristic Kα X ray beam produced in the dx layer can pass
through x depth and reach the detector producing an electronic pulse is:

P3 = e −µ.ρ0 . x / sen θ .ε Eq(12)

where:
µ = matrix mass absorption coefficient (cm2 g-1),
ε = detector efficiency for recording a photon of characteristic energy photons,
θ = take off angle (between the sample surface and the direction of the emergent bean.

The detector efficiency ε can be theoretically calculated from the data given by the manufacturer, the
distance between sample and detector and excitation conditions (vacuum, air or He). In this way some
authors [139, 143] calculate the detector efficiency for a Si(Li) for element from atomic number 13 (Al)
to 22 (Ti), using 55Fe as excitation source in vacuum, and from 19 (K) to 42 (Mo), using a 109Cd
source, in air (Figure 20).
EFICIENCY

ATOMIC NUMBER

Figure 20. Efficiency for a Si(Li) detector for different atomic numbers. From 13 (Al) to 22 (Ti) excitated
with 55Fe in vacuum. From 19(K) to Mo(42) with 109Cd in air [139, 143]

The fluorescent intensity dI due to a certain element in a dx layer can be written as following:

20
 ⎛ 1⎞
dI = G.e−µ0 .ρ0 .x / senθo .τ.⎜⎜1− ⎟⎟.w.f.ρ.dx.e−µ.ρ0 .x / senθ .ε Eq(13)
⎝ j⎠

Where G, called geometric factor, is proportional constant depending on the excitation-detection

geometry of the system, the intensity of the tube or activity of the source, etc. but independent of the
element.
The last equation can be written:

⎛ 1⎞
dI = G.e-(µ0 / senθ +µ / senθo ).ρ0.x.τ.⎜⎜1− ⎟⎟.w.f.ρ0.ε.dx Eq(14)
⎝ j⎠

The total mass absorption coefficient χ can be defined as:

χ = µ0 / sen θ 0 + µ / sen θ Eq(15)

And using Eq(8), it can be rewritten the equation 14 in the following way:

dI = G.ε.K.e − χ.ρ0 . x .ρ.dx Eq(16)

The integration of the Eq(16) on the base of the total thickness D gives the fluorescent intensity for a
given element:

1 − e − χ .ρ 0 . D
I = G.ε.K.ρ. Eq (17)
χ.ρ 0

The ratio ρ/ρo represents the “density” of the interest element (g element cm-3 sample) related to the
matrix density (g sample. cm-3 sample), therefore is the element concentration C in the sample as a
fraction of weight:

1 − e − χ .ρ 0 . D
I = G.ε.K.C. Eq (18)
χ
and considering

S = G.ε.K Eq (19)

Where S represents the sensibility of the spectrometer to the element, the Eq(18) can be rewritten:

21
 1 − e − χ .ρ 0 . D
I = S.C. Eq(20)
χ

Sometimes, this equation is expressed considering the surface density c (element g. cm-2 sample)
instead of the concentration C that is:

c = C . ρ o .D Eq(21)

and in this way the Eq(18) can be written in the following way:

1 − e − χ .ρ 0 . D
I = S.c. Eq(22)
χ.ρ o .D

The ratio in the equation 22 is called absorption factor for the given element:

1 − e − χ .ρ 0 . D
A= Eq(23)
χ.ρ o .D

In this way Eq(22) can be rewritten as:

I = S.c.A Eq(24)

Figure 21 shows a schematic representation of the dependency among these variables. As it was
mentioned in the beginning of this item, the model was calculated for the characteristic X-rays of the
K shell, that is the Kα line. In the same way can be obtained the same equations for the Lα lines
where the fundamental parameters will have other values.

PHYSICAL CONSTANTS

photoelectric fluorescent emition jump

coeficient yield intensity ratio

sensibility geometric detector

X-ray
factor eficiency
intensity

concentration

matrix absorption coefficient

incident energy
absorption Total absorption matrix absorption coefficient
factor factor characteristic energy

Figure 21. Schematic representation among variables in the fundamental X- ray equation for
monoenergetic excitation

22
It has to be remarked that in the case of thin thickness samples, the term χ.ρo.D tends to zero
therefore e-χ.ρo.D → 1, in this case the absorption factor is 1.

Thin thickness A=1 Eq(25)

In the opposite case for infinite thickness samples, the term χ.ρo.D → ∞, and e-χ.ρo.D → 0, in this case
the absorption factor has the following value:

1
Thick thickness A= Eq(26)
χ.ρ o .D

It is must be noted that if the intensity I given in Eq (22) and (24) for a given element is expressed in
counts per second and mA, sensitivity S will be expressed in cps g-l cm2 mA-1.
If the superficial density of the sample ρo.D is expressed in g cm-2 and the total mass absorption
coefficient in cm2 g-1.
The sensibility curve can be performed using thick or thin films from standard reference materials or
pure elements. In this case, the absorption factor can be calculated for these standards (Eq 23) and
with this intensity I value, can be calculated the elemental sensibility S by using the Eq (20) or (22)
(Figure 22).
SENSIBILITY (S)

ATOMIC NUMBER (Z)

Figure 22. Elemental sensitivity for atomic numbers between elements 13 (Al) to 22 (Ti), excited with 55Fe
in vaccumm and from 19 (K) to 42 (Mo) excited 109Cd in air [139, 143]

On the other hand, the elemental sensitivity S is related with the fundamental parameters K and the
detector efficiency ε by jeans a independent elemental constant called geometric factor G, as is
shown in Eq (16), and can be rewritten as:
S
G= Eq(27)
ε.K

23
This geometric factor must be constant for all energies and in this way it can be obtained an average
value which can be used for quantitative determinations.

4.- Quantitative and Qualitative Analysis

In XRF analysis, the energy of the emitted photons is about 1 to 30 KeV and in this case the auto-
absorption is very intense. This problem can be solved by dilution of the sample with a variety of
compounds as cellulose, boric acid, sugar, etc. and using the standard addition method.
In the case of samples containing few elements, such as alloys, it is possible to use multivariate
regression, where the calculation of the concentration of each element includes the intensity of the
rest of the elements beside the considered element [87, 174, 175] (Figures 23 y 24).
CONCENTRATION (%)

INTENSITY (.1000
INTENSITY (.1000 COUNTS)
COUNTS)

Figure 23. Quantitative analysis of Cr in stainless steel by EDXRF. Excitation: 109Cd) [87]
CONCENTRATION (%)

INTENSITY (.1000 COUNTS)

Figure 24. Quantitative analysis of Cu in alloy by WDXRF[87]

This method has the disadvantage in preparing a large number of standards to cover the elemental
concentration range in the samples. For instance, to analyze the concentration of n elements should be
prepared at least 2n-1 standards including each standard the n elements within concentration range
estimated.
Another option is to use the fundamental parameters method, based in a previous knowledge of
physical constants, geometric factor, detector efficiency, etc., where no calibration standards are

24
required, only pure elements are needed. The main disadvantage of this method is the calculation of
the sample absorption factor for each X-ray characteristic energy.
This can be bypassed in the semi-quantitative analysis, assuming the knowledge of the sample
fractions and considering samples with similar compositions. In this way, the minor element
determination in alloys can be achieved if the majority composition is known.
In the case of animal tissue samples, it is possible to calculate the average absorption factor for each
characteristic X-ray energy from estimated concentrations values of H, C, N, O, K, Ca, etc. from
literature.
In order to avoid this work, it is possible to use other methods on the basis of experimental
measurement of the absorption coefficient of the sample in those characteristic energies.
One of these methods is named irradiator method or the emission/transmission method[ 53, 139, 143 ].
Initially the irradiator is prepared with elements absent in the sample. The intensities of the
characteristic X-rays Ix of the present elements in the irradiator is measured, and immediately the
sample is placed between the irradiator and the detector, measuring again the intensities Ix of the
elements in the irradiator. Due to the presence of the sample, the original intensities Ix will diminish
due to the absorption of the sample and therefore it is possible to write:

I
R= = e − χ .ρ 0 . D Eq (28)
I0

where R represents the transmission of the electromagnetic radiation by the sample in the
characteristic X-rays energies in the irradiator one. Finally, by using an analytical balance, the
superficial density ρo.D of the sample can be determined and in this way to establish a relation
between the total absorption coefficient χ and the energy of X-rays for the analyzed sample.
Based on that relation, it is possible to calculate the absorption factors for the energies of the
elements present in the sample; and finally on the basis of elemental sensitivity, detector efficiency
and geometric factor, the elemental concentrations are determined [ 139, 143 ].
Another method, denominated backscatter radiation [26, 32], is based on the effect of incoherent
(Compton effect, with loss of energy) and coherent (Rayleygh effect, without loss of energy)
scattering. The intensities of these backscatter radiations depend on the average composition of the
matrix, being the incoherent radiation dispersed preferentially by the light fraction (low atomic
number elements, that is, from H to Si), therefore the coherent radiation is dispersed preferentially by
the heavy fraction (elements of atomic number upon Si). Based on the dispersed intensities, it is
possible to be estimate the light and heavy fractions of the matrix, and through the fundamental
parameters and iterative calculation, the elemental concentration in the sample can be estimated.

4.2.- Detection Limit

In an x-ray spectrum, a continuous line can be observed on the characteristic signal corresponding to
present elements in the sample. This line is mainly due to the interactions of radiations scattered by
the sample and also of the own characteristic radiations emitted by the elements. Thus, in signal is
composed by the true or net intensity of an element i plus a continuum or background (BG) in the
same position i.
The detection limit LDi (cps) for an element i is directly related with the BG intensity [3] in the same
position according to:

LD i (cps) = 3. BG i Eq(29)

25
In other words, this minimum detection limit can be expressed as an elemental concentration (µg g-1)
give by a net intensity equal to three times de BG intensity (cps):

3. BGi
LDi ( µg / g ) = Eq(30)
si
where
si is the elemental sensitivity

Acronyms
CGEN = Congresso Geral de Energia Nuclear, CNEN
ECPG/CENA/USP = Encontro Científico dos Pós-Graduados do CENA/USP
ENAN = Encontro Nacional de Aplicações Nucleares, ABEN/CNEN
ERAN = Encontro Regional de Aplicações Nucleares, ABEN/CNEN
SARX = Seminário Latino-Americano de Análisis por Técnicas de Rayos X
INAC = International Atlantic Nuclear Conference

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fluo-rescência de raios X, em suplementos minerais para animais. Tese, IPEN/CNEN - USP,
1994, 175 p. (orientador: V. F. NASCIMENTO FILHO).
[174] ZUCCHI, O. L. A. D., e V. F. NASCIMENTO FILHO - Caracterização qualitativa e
quantitativa de elementos, pela técnica de fluorescência de raios X, em suplementos minerais
para animais. Parte 1: Dispersão de comprimento de onda. Pesq. Agropec. Bras., Brasília, 30
(12): 1427-1439, 1995.
[175] ZUCCHI, O. L. A. D., e V. F. NASCIMENTO FILHO - Caracterização qualitativa e
quantitativa de elementos, pela técnica de fluorescência de raios X, em suplementos minerais
para animais. Parte 2: Dispersão de energia. Pesq. Agropec. Bras., Brasília, 30 (12): 1441-
1452, 1995
[176] ZUCCHI, O. L. A. D., e V. F. NASCIMENTO FILHO - Caracterização quali-quantitativa de
elementos em suplementos minerais para animais, pela técnica de fluorescência de raios X. In:
Anais do V CGEN, Rio de Janeiro, RJ, 1994, p. 853-857.

36
[177] ZUCCHI, O. L. A. D.; V. F. NASCIMENTO FILHO e S. M. SIMABUCO - Composición
química de suplementos minerales para animales, por el empleo de la técnica de fluorescência
de rayos X. Anais SARX’94, Punta de Tralca, Chile, 1994, publicado em 1996, p. 179-184.

Papers Published by the Author

1. SIMABUCO, S. M.; R. FIGUEROA, M. GARCÍA, e V. F. NASCIMENTO FILHO –
Comparação das concentrações de metais em águas potáveis das cidades de Córdoba, Temuco e
Campinas por ED-XRF. Anais do SARX´98, Huerta Grande/Córdoba, Argentina, 1998,
publicado em 2000, p. 31-36.
2. PARREIRA, P. S.; V. F. NASCIMENTO FILHO, C. A. PEREZ, e H. T. FUKUMA -
Determinação de tório e urânio em efluentes por TXRF com luz síncrotron. Anais do
SARX´98, Huerta Grande/Córdoba, Argentina, 1998, publicado em 2000, p. 43-48.
3. LOPES, G. N.; E. E. VALENCIA, e V. F. NASCIMENTO FILHO – Determinación de
elementos químicos presentes en pelos y plumas de animales salvajes en cautiverio por FRX
por dispersión en energías. Anais do SARX´98, Huerta Grande/Córdoba, Argentina, 1998,
publicado em 2000, p. 54–59.
4. SIMABUCO, S. M.; E. MATSUMOTO, e V. F. NASCIMENTO FILHO – Determinação de
metais pesados em águas naturais por fluorescência de raios X com radiação síncrotron. Anais
do SARX´98, Huerta Grande/Córdoba, Argentina, 1998, publicado em 2000, 84–89.
5. PARREIRA, P. S., e V. F. NASCIMENTO FILHO – Caracterização de fosfatos e fosfogesso
por fluorescência de raios X com fontes radioativas de 55Fe, 238Pu e 109Cd. Anais do VII CGEN,
Belo Horizonte, MG, 1999, 6 p. (CD-ROM).
6. SIMABUCO, S. M.; E. MATSUMOTO, e V. F. NASCIMENTO FILHO – Metals
preconcentration in natural waters by synchrotron radiation X-ray fluorescence analysis.
Activity Report of the Labotório Nacional de Luz Síncrotron/CNPq, Campinas, SP, 1997-98, p.
6.5-5 e 6.5-6 (publicado em 1999).
7. PARREIRA, P. S.; V. F. NASCIMENTO FILHO, C. A. PEREZ, e H. T. FUKUMA –
Determination of thorium and uranium in effluents by TXRF with synchrotron radiation.
Activity Report of the Laboratório Nacional de Luz Síncrotron/CNPq, Campinas, SP, 1997-98,
p. 6.6-7 e 6.6-8 (publicado em 1999).
8. APPOLONI, C. R.; F. R. ESPINOZA QUIÑONES, P. H. ARAGÃO, V. F. NASCIMENTO
FILHO, A. O. dos SANTOS, R. M. da CUNHA e SILVA, and P. F. BARBIERI - ED-XRF
study of Tupiguarani archaeological ceramics. Anais do VI International Conference on Non-
Destructive Testing and Micro-analysisis for the Diagnosis and Conservation of the Cultural
and Environmental Heritage (ART’99), Roma, Itália, 1999, p. 1327–1341.
9. ZUCCHI, L. A. D.; D. A. DIAS, V. F. NASCIMENTO FILHO, and M. J. SALVADOR –
Characterization of two medicinal plants by X-ray spectrometry. Journal of Trace and
Microprobe Techniques, New York, 18 (3): 441–450, 2000.
10. VIVES, A. E. S.; S. M. B. BRIENZA, V. F. NASCIMENTO FILHO, e S. M. SIMABUCO -
Disponibilidade de metais em material particulado em suspensão utilizando técnicas de
extração química e fluorescência de raios X. Anais do VI Congresso de Geoquímica dos Países
de Língua Portuguesa (VI CGPLP), Faro, Portugal, 2001, p. 556–560.
11. APPOLONI, C. R.; F. R. ESPINOZA QUIÑONES, P. H. A. ARAGÃO, A. O. dos SANTOS,
L. M. da SILVA, P. F. BARBIERI, V. F. NASCIMENTO FILHO, and M. M. COIMBRA -
EDXRF study of Tupi-Guarani archaeological ceramics. Radiation Physics and Chemistry,
Nova Iorque, 61 (3-6): 711- 712, 2001.

37
12. ALMEIDA, E., and V. F. NASCIMENTO FILHO - Determination of chemical elements in
biological samples using total reflection X-ray fluorescence technique. Anais do VIII CGEN e
V ENAN, Rio de Janeiro, RJ, 6 p., 2000, publicado em 2001, 6 p. (CD-ROM).
13. ESPINOZA QUIÑONES, F. R.; APPOLONI, C. R.;, P. H. A. ARAGÃO, A. O. dos SANTOS,
L. M. da SILVA, P. F. BARBIERI, V. F. NASCIMENTO FILHO, and M. M. COIMBRA -
EDXRF study of Tupiguarani archaeological ceramics. Anais do VIII CGEN e V ENAN, Rio
de Janeiro, RJ, 6 p., 2000, publicado em 2001, 8 p. (CD-ROM).
14. CARNEIRO, A. E. V.; S. M. B. BRIENZA, V. F. NASCIMENTO FILHO, S. M. SIMABUCO,
M. FAION, L. J. S. SOUZA, e W. F. SIQUEIRA – Emprego da fluorescência de raios X no
monitoramento de metais pesados em amostras de águas. Anais do VIII CGEN e V ENAN, Rio
de Janeiro, RJ, 2000, publicado em 2001, 6 p. (CD-ROM).
15. VALENCIA, E. P. E.; G. N. LOPES, e V. F. NASCIMENTO FILHO – Caracterização química
do material particulado fino em suspensão dos rios Piracicaba e Atibaia, SP, através da
fluorescência de raios X. Anais do VIII CGEN e V ENAN, Rio de Janeiro, RJ, 2000, publicado
em 2001, 6 p. (CD-ROM).
16. CUNHA e SILVA, R. M.; V. F. NASCIMENTO FILHO, C. R. APPOLONI, and C. A. PEREZ
– Analysis of archaeological ceramic using energy dispersive X-ray micro-fluorescence ( -
XRF). Activity Report of the Laboratório Nacional de Luz Síncrotron/CNPq, Campinas, SP,
2000, p. 33–34 (publicado em 2001).
17. KORNDÖRFER, C. M.; A. L. ABDALLA, I. C. S. BUENO, V. F. NASCIMENTO FILHO, E.
OWEN, e J. D. SUTTON - Estudo da cinética digestiva em ovinos alimentados com braquiária
e alfafa, usando a técnica de fluorescência de raios X. Veterinária Notícias, Uberlândia, 7 (2):
113–121, 2001.
18. ALMEIDA, E., and V. F. NASCIMENTO FILHO - Emprego da fluorescência de raios X por
reflexão total para a determinação da composição química em matrizes biológicas. Anais do
SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p. 37–43.
19. APPOLONI,C.R.; F. R. ESPINOZA QUIÑONES, P. H. A. ARAGÃO, A. O. DOS SANTOS,
L. M. da SILVA, P. F. BARBIERI, e V. F. NASCIMENTO FILHO - Estudo de cerâmicas
arqueológicas da tradição Tupi-guarani por fluorescência de raios X por dispersão de energia.
Anais do SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p. 24–30.
20. CUNHA e SILVA, R. M.; V. F. NASCIMENTO FILHO, E. P. E. VALENCIA, e E.
ALMEIDA - Determinação de Fe, Cu e Zn em aguardentes da região de Piracicaba, SP, pela
técnica de fluorescência de raios X por reflexão total. Anais do SARX´2000, São Pedro, SP,
Brasil, 2000, publicado em 2002, p. 240–245.
21. LOPES, G. N.; N. P. SAMPAIO, N. C. SILVA, H. T. FUKUMA, M. TOMAZELLO FILHO, e
V. F. NASCIMENTO FILHO – Estudo de fóssil arbóreo de Bonfim/RR - Brasil com MEV.
Anais do SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p.489–496.
22. MATSUMOTO, E.; S. M. SIMABUCO, C. A. PEREZ, e V. F. NASCIMENTO FILHO –
Análise de particulado atmosférico utilizando a reflexão total com radiação síncrotron. Anais
do SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p. 44–49.
23. PARREIRA, P. S.; V. F. NASCIMENTO FILHO, e H. T. FUKUMA - Determinação de tório e
urânio após digestão química utilizando a técnica de TXRF com tubo de mo e filtro “cutt-off”
com duplo refletor. Anais do SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p.
235–239.
24. PARREIRA, P. S.; V. F. NASCIMENTO FILHO, and H. T. FUKUMA - Phosphate rock and
phosphate fertilizers analysis using the TXRF technique after chemical digestion. Anais do
SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p. 246-252.

38
25. SALVADOR, M. J.; G. N. LOPES, V. F. NASCIMENTO FILHO, e O. L. A. D. ZUCCHI –
Controle de qualidade de chás comerciais por fluorescência de raios X. Anais do SARX´2000,
São Pedro, SP, Brasil, 2000, publicado em 2002, p. 144-149.
26. VALENCIA, E. P. E., e V. F. NASCIMENTO FILHO - Caracterização química de material
particulado fino em suspensão hidrotransportado no rio Piracicaba, SP, através da fluorescência
de raios X. Anais do SARX´2000, São Pedro, SP, Brasil, 2000, publicado em 2002, p. 150-157.
27. VIVES, A. E. S.; S. M. B. BRIENZA, V. F. NASCIMENTO FILHO, e S. M. SIMABUCO -
Determinação de metais pesados fracamente ligados em material fino particulado em suspensão
empregando a fluorescência de raios x com dispersão de energia. Anais do SARX´2000, São
Pedro, SP, Brasil, 2000, publicado em 2002, p. 13–18.
28. CUNHA e SILVA, R. M.; V. F. NASCIMENTO FILHO, C. R. APPOLONI, e C. A. PEREZ -
Caracterização química de fragmentos cerâmicos arqueológicos por microfluorescência de raios
X ( -XRF). Anais do I INAC, IX CGEN e VI ENAN, Rio de Janeiro, RJ, 2002, publicado em
2002, 4 p. (CD-ROM).
29. LOPES, F.; R. M. SILVA e CUNHA, V. F. NASCIMENTO FILHO, e C. R. APPOLONI –
Fluorescência de raios X por reflexão total aplicada à análise de elementos químicos em chá de
erva-mate (Ilex paraguariensis). Anais do I INAC, IX CGEN e VI ENAN, Rio de Janeiro, RJ,
2002, publicado em 2002, 4 p. (CD-ROM).
30. OLIVEIRA, H.; A. FURLAN, V. F. NASCIMENTO FILHO, e G. A. SARRIES - Composição
química de solo e biossólido pela técnica de fluorescência de raios X. Anais do I INAC, IX
CGEN e VI ENAN, Rio de Janeiro, RJ, 2002, publicado em 2002, 3 p. (CD-ROM).
31. MENEGÁRIO, A. A.; D. C. PELLEGRINOTTI, M. F. GINÉ, e V. F. NASCIMENTO FILHO
- Pré-concentração em sistema de fluxo para determinação multielementar em amostras de
águas fluviais por fluorescência de raios X com reflexão total. Anais do I INAC, IX CGEN e
VI ENAN, Rio de Janeiro, RJ, 2002, publicado em 2002, 6 p. (CD-ROM).
32. MENEGÁRIO, A. A.; A. C. F. GOMES, D. C. PELLEGRINOTTI, M. F. GINÉ, F. J. KRUG, e
V. F. NASCIMENTO FILHO - Remoção de interferentes em amostras com altos teores de
sólidos dissolvidos para a determinação de arsênico e selênio por TXRF. Anais do I INAC, IX
CGEN e VI ENAN, Rio de Janeiro, RJ, 2002, publicado em 2002, 5 p. (CD-ROM).
33. SALVADOR, M. J.; D. A. DIAS, V. F. NASCIMENTO FILHO, e O. L. A. ZUCCHI - Análise
elementar de Alternanthera marítima e Blutaparon portulacoides (Gomphreneae, Amaran-
thaceae) por fluorescência de raios X. Anais do I INAC, IX CGEN e VI ENAN, Rio de Janeiro,
RJ, 2002, publicado em 2002, 6 p. (CD-ROM).
34. VIVES, A. E. S.; S. M. B. BRIENZA, V. F. NASCIMENTO FILHO, e S. MOREIRA –
Emprego da fluorescência de raios X dispersiva em energia para avaliação da poluição por
metais pesados em amostras de água e sedimentos de lagos. Anais do I INAC, IX CGEN e VI
ENAN, Rio de Janeiro, RJ, 2002, publicado em 2002, 6 p. (CD-ROM).
35. BRIENZA, S. M. B.; A. E. S. VIVES, V. F. NASCIMENTO FILHO, e S. M. SIMABUCO –
Disponibidade de metais pesados em sedimentos de lagos utilizando técnicas de extração
química e de fluorescência de raios X. Anais do I INAC, IX CGEN e VI ENAN, Rio de
Janeiro, RJ, 2002, publicado em 2002, 5 p. (CD-ROM).
36. BARRETO, S. R. G.; W. J. BARRETO, J. NOZAKI, V. F. NASCIMENTO FILHO, e P. H. A.
ARAGÃO - Identification of metals in bordering soils and sediments of lake Ipê-MS, Brazil, by
EDXRF technique. Anais do I INAC, IX CGEN e VI ENAN, Rio de Janeiro, RJ, 2002,
publicado em 2002, 2 p. (CD-ROM).
37. SOLCI, M. C.; A. F. PELICHO, P. H. A. ARAGÃO, e V. F. NASCIMENTO FILHO -
Aplicação da fluorescência de raios X na determinação de componentes insolúveis nas águas de
precipitação em região continental subtropical. Anais do I INAC, IX CGEN e VI ENAN, Rio
de Janeiro, RJ, 2002, publicado em 2002, 4 p. (CD-ROM).

39
38. ARAGÃO, P. H. A., E. DI MAURO, N. M. SUGUIHIRO, L. G. L. RAMPAZO, M. A. C.
MELO, A. PAESANO JUNIOR, e V. F. NASCIMENTO FILHO - Estudo de fitopatologias
através da técnica de fluorescência de raios X por dispersão de energia. Anais do I INAC, IX
CGEN e VI ENAN, Rio de Janeiro, RJ, 2002, publicado em 2002, 5 p. (CD-ROM).
39. BRUNO, R. L.; A. F. ALMEIDA , J. A.GONÇALVES, V. F. NASCIMENTO FILHO, e J. R.
COURY - Concentração de material particulado fino, PM-10, e super fino, PM-2,5, presentes
na atmosfera na cidade de São Carlos e suas possíveis fontes. Anais do XXX Congresso
Brasileiro de Sistemas Particulados (XXX ENEMP), realizado em São Carlos, SP, no período
de 20 a 23/out/2002, 10 p. (CD-ROM), 2002, publicado em ago/2002).
40. ALMEIDA, E.; V. F. NASCIMENTO FILHO, E. P. E. VALENCIA, and R. M. CUNHA e
SILVA – Concentrations of Fe, Cu and Zn in rum by EDXRF using APDC pre-concentration.
Journal of Radioanalytical and Nuclear Chemistry, Budapest, 252 (3): 541–544. 2002.
41. MATSUMOTO, E.; S. M. SIMABUCO, C. A. PEREZ, and V. F. NASCIMENTO FILHO -
Atmospheric particulate analysis by synchrotron radiation total reflection (SR-XRF). X-Ray
Spectrometry, Londres, 31 (2): 136-140, 2002.
42. ZUCCHI, O. L. A. D.; V. F. NASCIMENTO FILHO, and H. SALVIO NETO – Application of
X-ray fluorescence to the determination of metals in commercial tablets containing digoxin.
Journal of Trace and Microprobe Techniques, Nova York, 20 (1): 145–149, 2002.
43. SALVADOR, M. J.; G. N. LOPES, V. F. NASCIMENTO FILHO e O. L. A. D. ZUCCHI –
Quality control of commercial tea by X-ray fluorescence. X-Ray Spectrometry, Londres, 31
(2): 141–144, 2002.
44. MENEGÁRIO, A. A.; D. C. PELLEGRINOTTI, M. F. GINÉ, and V. F. NASCIMENTO
FILHO - On-line preconcentration flow system for multi-elemental analysis by total reflection
X-ray fluorescence spectrometry. Spectrochimica Acta, Part B, 58 (3): 543–549, 2003.
45. ARAGÃO, P. H. A., E. DI MAURO, N. M. SUGUIHIRO, L. G. L. RAMPAZO, M. A. C.
MELO, A. PAESANO JUNIOR, e V. F. NASCIMENTO FILHO - Estudo de fitopatologias
através da técnica de fluorescência de raios X por dispersão de energia. Revista Brasileira de
Pesquisa e Desenvolvimento, Rio de Janeiro, 4 (3, parte 1): 625-629, 2002, publicado em
2004.
46. CUNHA e SILVA, R. M.; V. F. NASCIMENTO FILHO, C. R. APPOLONI, e C. A. PEREZ -
Caracterização química de fragmentos cerâmicos arqueológicos por microfluorescência de raios
X (m-XRF). Revista Brasileira de Pesquisa e Desenvolvimento, Rio de Janeiro, 4 (3, parte
1): 975 - 978, 2002, publicado em 2004.
47. LOPES, F.; R. M. SILVA e CUNHA, V. F. NASCIMENTO FILHO, e C. R. APPOLONI –
Fluorescência de raios X por reflexão total aplicada à análise de elementos químicos em chá de
erva-mate (Ilex paraguariensis). Revista Brasileira de Pesquisa e Desenvolvimento, Rio de
Janeiro, 4 (3, parte 1): 999-1002, 2002, publicado em 2004.
48. SALVADOR, M. J.; D. A. DIAS, V. F. NASCIMENTO FILHO, e O. L. A. ZUCCHI - Análise
elementar de Alternanthera marítima e Blutaparon portulacoides (Gomphreneae, Amaran-
thaceae) por fluorescência de raios X. Revista Brasileira de Pesquisa e Desenvolvimento, Rio
de Janeiro, 4 (3, parte 1): 1136-1140, 2002 (publicado em dez/2004).
49. VIVES, A. E. S.; S. M. B. BRIENZA, V. F. NASCIMENTO FILHO, e S. MOREIRA –
Emprego da fluorescência de raios X dispersiva em energia para avaliação da poluição por
metais pesados em amostras de água e sedimentos de lagos. Revista Brasileira de Pesquisa e
Desenvolvimento, Rio de Janeiro, 4 (3, parte 2): 1163-1168, 2002 (publicado em dez/2004).
50. BRIENZA, S. M. B.; A. E. S. VIVES, V. F. NASCIMENTO FILHO, e S. M. SIMABUCO –
Disponibidade de metais pesados em sedimentos de lagos utilizando técnicas de extração
química e de fluorescência de raios X. Revista Brasileira de Pesquisa e Desenvolvimento,
Rio de Janeiro, 4 (3, parte 2): 1169-1173, 2002 (publicado em dez/2004).

40
51. BARRETO, S. R. G.; W. J. BARRETO, J. NOZAKI, V. F. NASCIMENTO FILHO, e P. H. A.
ARAGÃO - Identification of metals in bordering soils and sediments of lake Ipê-MS, Brazil, by
EDXRF technique. Revista Brasileira de Pesquisa e Desenvolvimento, Rio de Janeiro, 4 (3,
parte 2): 1188-1198, 2002 (publicado em dez/2004).
52. CUNHA e SILVA, R. M.; E. ALMEIDA, and V. F. NASCIMENTO FILHO - Determination of
Fe, Cu and Zn in sugar-cane spirits commercialized in the Southeastern Brazil by TXRF.
Journal of Radioanalytical and Nuclear Chemistry, Budapest, 260 (1): 3–7, 2004.
53. GOMES, A. C. F.; A. A. MENEGÁRIO, D. C. PELLEGRINOTTI, M. F. GINÉ, and
NASCIMENTO FILHO, V. F. – A hydride generation flow system for determination of arsenic
and selenium by total reflection X-ray fluorescence. Spectrochimica Acta, Part B, 59 (9):
1481–1484, 2004.
54. BARRETO, S. R. G.; J. NOZAKI, E. OLIVEIRA, V. F. NASCIMENTO FILHO, P. H. A.
ARAGÃO, I. S. SCARMINIO, and W. J. BARRETO – Comparison of metal analysis in
sediments using EDXRF and ICP-OES with the HCl and Tessie extraction methods. Talanta,
Amsterdam, 64 (2): 345–354, 2004.
55. BRIENZA, S. M. B.; A. E. S. VIVES, V. F. NASCIMENTO FILHO, e S. MOREIRA –
Variabilidade sazonal de metais em amostras de água, material fino particulado em suspensão e
sedimento de fundo. Anais do SARX´2002, Nova Friburgo, RJ, Brasil), 6 p. (em publicação).
56. CUNHA E SILVA, R. M.; F. LOPES, e V. F. NASCIMENTO FILHO - Análise de K, Ca, Fe,
Cu e Zn em fumaça de incenso pela técnica de fluorescência de raios X. Anais do SARX´2002,
Nova Friburgo, RJ, Brasil), 6 p. (em publicação).
57. GOMES, A. C. F.; A. A. MENEGÁRIO, D. C. PELLEGRINOTTI, M. F. GINÉ, e V. F.
NASCIMENTO FILHO – Determinação de arsênio e selênio em amostras de águas por TXRF
após pré-concentração e separação da matriz através da geração de hidretos em sistema de
fluxo. Anais do SARX´2002, Nova Friburgo, RJ, Brasil), 6 p. (em publicação).
58. OLIVEIRA, H.; R. M. CUNHA e SILVA, C. A. PEREZ, A. FURLAN, J. MORTATTI, e V. F.
NASCIMENTO FILHO - Composição elementar em sedimentos de rio próximos a descargas
de resíduos domésticos por EDXRF com excitação por radiação síncrotron. Anais do
SARX´2002, Nova Friburgo, RJ, Brasil), 6 p. (em publicação).
59. SIMABUCO, S. M; E. F. O. JESUS; R. T. LOPES; C. PEREZ; V. F. NASCIMENTO FILHO;
E. MATSUMOTO; R. S. S. COSTA; M. G. DO CARMO TAVARES, and C. SAUNDERS -
Total reflection X-ray fluorescence with synchrotron radiation applied to biological and
environmental samples. International Symposium on Utilization of Accelerators/IAEA, paper
IAEA-SM/355-157, 10 pag., 2001 (submetido).
60. OLIVEIRA, A. L.; E. ALMEIDA, F. B. R. da SILVA, e V. F. NASCIMENTO FILHO - Exotic
Brazilian tropical fruits: elemental content evaluated by energy dispersive X-ray fluorescence.
Scientia Agricola, Piracicaba, 9 pag., set/2004 (submetido).

Virgilio F. Nascimento Filho

Nuclear Instrumentation Laboratory
Nuclear Energy for Agriculture Enter (CENA)
Univertsity of Sao Paulo (USP). Sao Paulo Brasil
virgilio@cena.usp.br

41
 2
Sample Preparation

Graciela Custo, Dora V. de Leyt and Cristina Vázquez

1.- Introduction
Sample preparation is one of the most important steps in all analytical determinations whatever
technique is going to be used. Analytical results are good in keeping with the rate which sample
preparation procedures have been done adequately selected.
In the particular case of X-Ray Fluorescence (XRF) automatic sequential modern spectrometer can
analyze up to 20 element in a few seconds, then sample preparation is the time limiting step in the
overall analysis1-9, except the case that sample can be analyze without any special preparation. This
case makes XRF spectrometry the most versatile of all instrumental analytical methods. Provides
qualitative information, quantitative multielement analysis, simple interpretation of the spectrum
and in most cases is a nondestructive technique.
It has seen several changes in both instrumentation and specimen preparation equipment over the
years. As computers have advanced it have gone from a basic matrix to the more advanced
fundamental parameter procedures utilized by equipment manufacturers today.
Conventional XRF is applicable to all chemical elements from atomic number 4 (beryllium) to 92
(uranium), in a wide range of concentration: 100% to 1 µg/g in solids and 0,1 µg/mL in liquids.
The sensibility is:
20 µg/g – 1000 µg/g Z ≤ 21 (Ti)
5 µg/g - 1 0 µg/g up to Z = 42 (Mo)
1 µg/g Z >42 Mo

42
The sample preparation can be physical, chemical or both. In X-ray analysis one the main
objectives is to minimize or to eliminate matrix effect and particle heterogeneity. One of the most
important forms is by means of the dilution.
Previous to the analysis several important aspects have to take in to account
• Processing a sample always contaminates it. Successful analysis depends on recognizing
the sources of contamination and controlling them.
• To have a previous knowledge of the sample
• How many and which analytes have to be analyzed
• Selecting the appropriate excitation conditions
• Choosing the best line to use for the quantitative analysis
• The concentration of the analyte in the sample (mg/kg, mg/g, g/100g, mg/l, mg/ml)
• Type of matrix (all element plus analyte)
• Required precision and accuracy
Analyzed samples by XRF fit into three main categories:
1) Samples, which can be, handled directly following some simple pre-treatment such as
pelletizing or surfacing. For example, homogeneous samples of powders, bulk metals or
liquids.
2) Samples requiring significant pre-treatment. For example, heterogeneous samples, samples
requiring matrix dilution to overcome inter-element effects and samples exhibiting particle
size effects.
3) Samples requiring special handling treatment. For example, limited size samples, very low
analyte concentration or previous analyte separation, etc.
Because of the wide scope of sample that can be analyzed by XRF, it is better to study sample
preparation related with the different specimen presentation, and this term will be used to indicate
the physical form, which the specimen is placed in the X-ray spectrometer.
Table 1 shows previous classification according to the physical state of the sample.

Solids Liquids (organic or inorganic) Gases and vapors

Bulk Solution They may be taken up in
Turning Suspension solution or in solid absorber
chip
drilling
shot
shaving
Granule
Powder
Fusion products
Coatings
Films, platings

Table 1. Classification of samples by physical state

43
Another useful classification is by the field of application:
Metals and alloys
Minerals and ores
Glasses and ceramics
Plastic, rubber, wood
Food
Biological: tissues, fluids, etc.
Textile products
Environmental
Continuous on-stream analysis and process control

2.- Samples Requiring No Treatment

In the case of purely qualitative analysis is sufficient that the sample fit in the chamber of the
spectrometer. For portable equipment this condition is not necessary because does not exist a
chamber. It is better to give a little surface treatment in order to improve the response of the
signals.
For quantitative analysis two problems are of particular importance. First, it is necessary that all
samples and standards cover the same irradiated area of the sample cell and second, all samples and
standards must have a roughly flat surface. In routine analysis the first of these problems can be
avoided using a mask that gives the same irradiation area.
The problem of giving all samples a surface finish of sufficient fineness depend of the material, for
metals they have to be polish and for powders they have to press into a pellet, these subject will be
studied in the next section.

3.- Equipment for Sample Preparation

Laboratories that manage full range XRF applications should have complete equipment set for
sample preparation. The following list shows the basic equipment required:
Rotating polishing wheel: abrasive papers and cloths
Abrasive diamond paste
Mixer/mill with plastic and tungsten carbide balls, with a set of vials of different materials (plastic,
agate, steel, etc.) and sizes
Standardized sieves of different materials (plastic, cupper, steel, etc.)
Hydraulic press (manual or motorized) for pressures up to 7000 Kg/cm2
Mortars and pestles: agate, tungsten carbide, steel, etc.
Pellet dies set of various sizes made of hardened stainless steel.
Oven
Muffle furnace
Crucibles (Pt, Pt-Au, graphite), casting mold, forceps with Pt tip
Microwave digestion equipment
Analytical and microanalytical balance

44
Filtration apparatus
Membrane filters of different materials (cellulose, Nuclepore®, etc.)
Ion-exchange columns
Specifics elements for XRF analysis have to be added: various sizes and materials of specimen
cells, mounting devices and masks according to different specimen presentations (Figure 1)
In addition, for TXRF sample preparation is necessary:
Bench laminar flow in a clean area
Infrared lamp
Reflectors (Perpex®, quartz, etc.)
Sub-boiling distillator
Deionized distilled water from Millipore Milli Q (resistivity = 18.0 MΩ. cm)
Adjustable and fixed volume pipettors with corresponding disposable tips

Figure 1: Cell samples and masks made of aluminum, plastic and Teflon®

4.- Bulk Solid Samples

Samples may be metal, ceramic, glass, plastic, rock, mineral, etc.; and have different presentations:
ingot, bar, rod, tube, sheet, or fabricated parts. Solid samples can be reduced to powders, briquets,
solutions, or supported specimens by using specific procedures after described. Figure 2 shows
different kind of bulk samples.

Figure 2. Different sample presentation: powder, shot, bulk, shavings and small-fabricated parts (from
left to right)
4.1.- Metals

45
Surface preparation is very critical considering this process homogenizes grain size of any
constituent between samples and standards. The final polished surface could be made by using
manual or mechanic methods starting by coarse to fine polishing.
Five stages in surface preparation can be considered10:
Stage 1: Coarse grinding
Stage 2: Mounting
Stage 3: Fine grinding
Stage 4: Rough polishing
Stage 5: Final polishing
Coarse grinding: this step is particularly important is the specimen has been removed from the
bulk by destructing techniques. It may be performed on high-speed disc grinders, which have high
material removal rates and maintain flatter sample surfaces.
Mounting provides a safe, convenient means of supporting samples for either manual or automatic
polishing. This step is specially recommended for small and thin samples. In these cases, samples
must be previously included in acrylic resins before polishing.
Fine grinding: the purpose of this step is to reduce the overall level of surface deformation. Using
polishing machine or manual with silicon carbide abrasive papers may perform it. A typical
sequence of 240, 320, 400 and 600 grit abrasive paper is used.
Rough polishing: is a very important step to maintain specimen flatness. It may be done by using
diamond paste compounds (15 to 1 µ) deposited on low or no napped cloths. It is a good practice to
spend more time on rough polishing and less on final polishing.
Final polishing: this step must be properly performed and will reveal the true microstructure if
rough polishing has been accurately carried out. Aluminum oxide, more often referred to as
Alumina (0.05 µ sizes), is the most popular final polishing abrasive. Two different crystalline
structures are available: the alpha and the gamma form been recommended the latter one because is
harder. Napped cloth is generally used for this step.
The use of lubricants is strongly recommended in all steps considering they allow the material stick
removal on the abrasive foil. Table 2 shows more common abrasive and corresponding lubricants.
Figure 3 (a) shows a semi-automatic standard polisher, which consist in a carefully balanced
bronze wheel fitted directly to the specially constructed dual-speed motor, and (b) two samples
with their surfaces polished following the all procedure above described.

Abrasive Lubricant Lap or Wheel covering

SiC (sand plus coke) Water Carbimet™
Al2O3 (synthetic fusion of bauxite) Water Carbimet™
MgO Water Microcloth™
Boron Carbide Water Disc grinder
Diamond powder Oil Nylon cloth

Table 2. Abrasive and corresponding lubricants for metal surface preparation

46
 (a) (b)
Figure 3. (a) Polishing with a semi-automatic polisher using SiC as abrasive and water as lubricant, (b)
Finished surface in a bulk and a fine foil of metal sample included in acrylic.

4.2.- Powders
XRF have the ability to analyze many different types of powder samples with several different
types of specimen preparation, for this kind of sample presentation, there are many available
preparation techniques.
This first stage of the analysis must be done carefully, with a suitable treatment selection to avoid
the introduction of any type of error that will carry in later stages. Errors up to 80% can be
attributed to an incorrect sample preparation, nullifying all later consideration such as calculation
of the matrix effects, data processing and evaluation of the results.
In the case of geological samples the preparation begins in the sampling site. Sampling
methodology should assure the representativeness of the final portion of the specimen. Therefore it
is convenient that the analyst knows history previous of the sample and the steps from the sampling
to its reception at the laboratory in order to interpret possible anomalies in the results.
When samples are received as powder, treatments of cutting, crushing, milling, sifting, etc could be
previous done introducing potential sources of error. The sample can be contaminated with the
sampling devices. In this case, the portion of the sample in direct contact with the device has to be
discarded. Sampling devices can be decontaminated between successive samples to avoid cross
contamination by immersing the samplers in plastic pail containing water and the wiping them with
a paper towel.
The packing used to contain the sample also must be considered, preferring impermeable plastic
materials. The paper packing can contain Hg, S, P, Ca, Ti and some ink used for the identification
can contaminate with Fe.
Vehicles used for transportation the samples to the laboratory can be a potential source of
contamination if they are not suitably conditioned (e.g., oxidized, dirty, wet surfaces, etc.).
Vibrations of the vehicle during the transport cause heaviest minerals go to the bottom producing
heterogeneities. During transportation, samples have to be handled in the same orientation in which
they were sampled with the appropriate markings on the container.
For storage, the sample field bags have to be protected from extreme heat, cold and moisture. In
order to maintain sample integrity, it is recommended to seal bags with a parafilm for a long
storage period (in excess of five months).
Some factors can affect the precision and accuracy of the analysis. Among them we can mention:
particle size effect, density variations between samples, non-reproducible roughness surfaces and
heterogeneity. These effects become more critical for light elements (Z <21 =Ti). For

47
heterogeneous samples, errors can reach more than 30% and in the case of particle size effect,
errors can exceed 50%. The matrix effects also lead to errors about 80% if they are not considered.
Sample must be grounded totally and once, if this is not possible, will have to be divided in suitable
portions, grinding each one and joining all partial portions homogenizing, quartering and finally
taking a representative portion from the total.
Figure 6 shows the different forms in which a powder sample can be analyzed; the chosen way will
depend on the atomic number of the element, concentration, matrix, accuracy, precision and time in
which the result is required.

(a) (b)
Figure 4: Mortars (a) mortar agate and a plastic vial with 2 plastic balls for mixing, (b) diamonite
mortar (a structural ceramic material made primarily from alumina)

Grinding
Bulk sample Powder sample

Checking size particle

Acceptable yes Analysing as

No size powder

Melting with a flux and molding

yes

Analysing as a glass disk

Briqueting
Grinding and briqueting
Analysing as a
pellet

Figure 6. Different way to analyze solid samples

48
4.2.1. -Control of Reproducibility
The control of the reproducibility in the sample preparation methods is the first step that has been
done in all analysis.
After verifying that the steps followed for the obtained sample lead to a reasonable standard
deviation for the analysis it will start with the use of the standards reference for the calibration
curve. If the calculated deviation for the method of sample preparation (Sm) is not satisfactory, it
has to control the instrumental variations (Si) in order to discard or not probable source of deviation
(Figure 7)

SAMPLE

10 specimens take one at random

measure once each sample measure 10 times

Sm Si

Sm compare with Si

Figure7. Scheme for checking the sample preparation procedure

4.2.2.- Presentation
4.2.2.1.- Loose powders
It is the easiest and fastest procedure when great accuracy and precision is not required. In order to
carry out the analysis, the powder is directly placed into the sample cell that consist in an open
cylinder cover on the bottom with a suitable thin film secured with a ring or collar. The choice of
the film material depends on the specific application. The most common materials are: Mylar,
polycarbonate, polypropylene and Kapton. The following requirements may be taken into account:
• strength: leakage or rupture of the film could cost damage to spectrometer
• transmission: This property is very important for light elements. Polycarbonate film 0.20
micron is considerably better for the lighter elements.
• Purity: Mylar film is suitable for most analysis, it may contain trace levels of Zn, P, Ca and
Sb. Polypropylene may contain Al, Ti, Fe, Cu and SiO2. Kapton film is free of all metallic
impurities.
• Chemical attack: this property is especially important in the case of liquid samples. It is
recommended testing the film by exposure to the sample for several minutes before
running time in the spectrometer.
It must be noted that enough powder were poured into the cell to exceed the infinite thickness.
Working with loose powders make difficult to maintain the sample preparation reproducibility
among samples and standards due to different packing.

49
Loose powder analysis permits to employ standard addition and internal standard methods. In this
case, particles size of both, sample and standard must be equivalent, homogenizing components
adequately.
4.2.2.2.- Briquets
Briqueting technique is applicable to powders from different materials: rocks, sand, soils, plants,
roots, cements, paper, cloth, metal granule, chips, shot, drillings, wire, etc1-2, 12-14.
The powder is compacted or pressed into a self-supporting pellet. If the sample powder is not easily
compressed into stable and cohesive pellet, it is necessary to admixture it with a binder. The most
recommended are: starch, microcrystalline cellulose, high molecular weight polyalcohols (moviol,
polyviol, etc), boric acid, wax, borax, sugar, urea, carbon, etc. In all cases, high purity binder
materials have to be used, besides it may be non-hygroscopic and particle size and density similar
to the sample.
Sample/binder relationship can vary in a wide range depending on the composition of the sample.
In order to assure an efficient mixing, the volume of the vial has to be filled 1/3 approximately
including adequately balls. On the other hand, its presence increases the background radiation due
to lighter matrix absorbs the characteristic X ray radiation of the analyte increasing detection limits.
The most convenient way to obtain a pellet is by briqueting at high pressure in a press using a die
set (Figure 8).
The advantages of working with briquets are the following:
1. Much easier to handle and conserved.
2. Control of some parameters such as packing density and thickness.
3. Possibility of doing the measurements in vacuum without placing Mylar foil to maintain the
sample
4. Improving the characteristic X ray radiation of the analyte respect to the loose powders.

Figure 8. Die set of different sizes

4.2.3.- Minimizing Particle Size Effect

In powders samples, the intensity of the analyte line may be affected by the particle size and
particle size distribution even if the composition of the sample is homogeneous. Segregation may
occur when the particle size of the powder is not uniform or when particles have different densities1
Generally, the fluorescent intensity increases when particle size decreases, except for matrices
highly absorbents; this effect is more critical for elements with atomic number less than 21.

50
Reducing the particle size of the sample minimizes particle size effect. For semi quantitative
determinations, 50 µm size produces satisfactory results especially for elements with atomic
number more than 25. For quantitative determinations is recommended 10µm or less.
One or more of the following methods minimizes particle size effects: grinding, briqueting, glass-
disk obtaining by fusion.
4.2.3.1.- Grinding
Materials for mortars have to be chosen according with the composition of the sample. The
operation can be made manual or mechanically. An excessive milling can cause absorption or loss
of H2O, CO2, and S and also oxidize Fe2+ to Fe3+; in addition, contamination with the equipment
can occur. Mortars must be scrupulously cleaned by grinding a portion of the sample and discard it
when the amount of sample is enough. In order to facilitate the grinding some drops of alcohol,
acetone or another volatile solvent could be added. Table 3 shows, as example, used typical
material and its characteristics for grinding.

Typical Composition
Major Minor elements Resistance Comparative
elements Efficiency
Material Hardness to abrasion Durability
Hardened Fe Cr, Si, Mn, C MOHS 5½-6 Moderate High High
Steel

Zirconia Zr, Y Hf MOHS 9 Very High High High

Stainless Fe, Cr Ni,Mn1, S1 MOHS 5-5½ Moderate High High

Steel

Tungsten W, C, Co Ta, Ti, Nb MOHS 8½+ High Long wearing but Very High
carbide subject to
breakage

Alumina Al Si, Ca, Mg MOHS 9 Very High Long-wearing, but Moderate

ceramic brittle

Agate Si Al, Na, Fe, K, MOHS 6-7 Extremely Very long- Moderate
Ca, Mg2 High wearing, but
brittle

Boron B, C - MOHS 9½ Very High Very long- N/A

Carbide wearing but brittle

Plastic C - MOHS 1½ Low Low, but Low for

disposable grinding

1 2
Possibly Present All reported less than 0.02%

Table 3. Materials for mortars and its characteristics

51
After grinding, particle size is controlled by sifting the pulverized material through a series of
sieves with known mesh size. Sifting process produces heterogeneity due to the sample separation
in portions of different grain size. This can be avoided grinding again the portion containing the
bigger size of the particles. Finally sample portions have to be homogenized exhaustively. Silk or
nylon sieves are preferred to bronze, cupper or steel, since these last ones produce contamination
with Pb, Cu, Zn, Fe, Ni, Cr and Mn. The inconvenient to use the formers is the electrostatic charge
produced when the powder is sifted (Figure 8). Table 4 shows mesh size equivalents expressed in
microns and inches.

Figure 8. Nylon, copper and steel sieves (left to right)

Mesh Microns Inches

20 840 0.0328
50 297 0.0116
100 149 0.0058
120 125 0.0049
140 105 0.0041
170 88 0.0034
200 74 0.0029
230 63 0.0025
270 53 0.0021
325 44 0.0017
400 37 0.0015
625 20 0.0008
1250 10 0.0004

Table 4. Mesh size equivalents in microns and inches

4.2.3.2.- Fusion Method

In 1956 Claisse proposed the techniques of fusion as an alternative for the transformation of
powders samples into a glass-disk.
Fusion method is most often used for samples, which are hard to dissolve in acid, difficult to
prepare as homogeneous pressed powders or both 1-9, 15-18.

52
Fusion methods have many advantages regardless of whether the fusion product will be used as a
glass, grinded or briqueted.
1. Eliminate particle size effects
2. Eliminate the mineralogical effects
3. The matrix effects disappear in greater or smaller extension according to the dilution.
4. The addition of internal standard or absorbent substances is made easily and the mixture
obtained after the fusion is perfectly homogenous.
5. The surface texture is smooth and uniform
6. The standards are very convenient stored
7. Properly prepared disks undergoes no significant change after 20 h of irradiation
The disadvantages of the fusion techniques are:
1. Characteristic x ray intensity for low Z elements are reduced by dilution and absorption, and it
is worst f in case of trace and minor constituents
2. Considerable time and effort are required
3. Glass disk may be break in storage due to the stress or devitrification
Fusion Technique. The samples are pulverized, if is necessary, and mixed with a flux in an
appropriate ratio (to 1:2 from 1:20). This mixture is heated until flux melts in an inert, heat resistant
crucible. Heating is maintained, and the crucible agitated. The sample dissolves in the flux or
reacts, yielding a clear and homogeneous melt. The melt is poured into a mold and cooled slowly,
finally a glass disk of a single phase is formed.
It is possible to fuse samples manually with simple equipment, or using an automated,
programmable, multi-burner fluxer.
Fusion technique is very versatile. The most important variables are:
1. Type of flux: it depends on the material that constitutes the sample. The flux can include: glass
maker agents, not wet agents and oxidizing agents.
2. The sample/flux rate
3. The internal standard addition
4. The addition of a heavy component to obtain an absorbent matrix.
Table 4 shows the most used fluxes and their temperature of fusion.
The flux more used is the alkaline borates, lithium tetraborate (Li2B4O7), lithium metaborate
(LiBO2) or a mixture of the two.
Lithium tetraborate is recommended for samples that contain a high basic oxide concentration such
as calcites and dolomites.
Lithium metaborate is more suitable when the sample contains a high acid oxide concentration,
obtaining melts are more fluid, and it is used for silicate and refractory samples. These flux
dissolve almost anything except metals, sulfides, and organics. Fluxes with lower melting points
than lithium borates are sodium tetraborate (Na2B4O7) and potassium pyrosulfate (K2S2O7), used
for more specialized applications. A mixture of both is recommend for samples containing silicon
oxide or aluminum oxide as greater constituent.
It is not easy to establish general rules with respect to the use of the lithium tetraborate or sodium
tetraborate, both are consider when the working temperature is high (1100-1200 0C).
The addition of lithium carbonate or lithium fluoride increases the basicity or acidity respectively,
besides decreases the temperature of fusion, increases the reaction speed and the fluidity of the

53
melt. When the sample to analyze contains light elements are better to use the lithium salt instead
of the sodium due to the lower x-rays absorption.

Flux Melting Point

Li2B4O7 920 OC
LiBO2 845 OC
Na2B4O7 740 OC

Table 4. Fluxes and melting point

Heating and Moulding. The crucibles more used are platinum; platinum + 5% gold and graphite.
The platinum crucibles have the disadvantage that the samples are usually adhered to the walls, and
it is only given off by fusion with sodium carbonate. The platinum crucibles + 5% gold have the
advantage of which the adhesion is much smaller. The graphite crucibles are much cheaper but
contaminate the sample, the using must be limited to samples without sulfides or sulphates.
Times of fusion oscillate between 5-30'. The fusion can be made using: Mecker burner, electrical
muffle or induction with high frequency. Agitation during the fusion is very important for the
homogenous glass production.
A polished glass disc can be obtained by moulding in a circular mold (same material as the
crucible) of the adequate size according to the spectrometer.
A good glass disc must be: transparent, uniformed in color and thickness, must have at least one
polished surface to flat mirror and some analysts prefer to grind the disc and to press the obtained
dust. With a good glass disc procedure is not necessary; it would only be useful if the glass were
not homogenous; then the fusion is used to produce small particles; the glass ground gives the
necessary homogeneity and the pressed produces a reproducible surface.
General Considerations. Temperature of the mold is very important; if it is very cold or very hot
the glass disc could be adhered or cracked.
The amount of tensions in the glass disc is function of the rate of cooling. The tensions do not
affect the result but increase the probability of cracking.
A rough surface of the mold makes difficult the removing of the glass disc and usually it cracks
when cools off.
Figure 9 shows two crucibles made of Pt and two different size Pt-Au molds for making glass disks
manually.

Figure 9: Pt crucibles, Pt-Au molds and glass disk

54
4.2.4.- Airborne Particulate Matter
In this kind of sample generally a small piece of material is available. The collected sample may be
prepared into thin layers and measured directly or put in solutions or suspension.
The aerosol consists of a complex and interacting unstable dynamic system. More than 50 % of all
air pollutants are preferentially present in particulate matter rather in gas.
The elemental analysis of bulk or size fractionated air particulate matter represents a unique
problem. May elements in widely differing concentration and atomic number are of interest. The
total amount of sample material is usually very small while often many samples have to be
analyzed.
There are several techniques for sampling airborne particulate matter: wet dry impaction, thermal
and electrostatic precipitation, centrifugation and filtration. Filtration of a known volume of air
through a fibrous or membrane type filter is the most widely used technique for sampling aerosols
in view of analysis by XRF19.
Filter collection systems can only be used for the determination of the total ambient concentration
of the elements. They have no capability of resolving the particle size distribution.
Of the collection system with a capability of size classification, inertial impactors are most popular.
The application of XRF is less simple in this application because of the considerably smaller
amounts of material, which is sampled.
Available aerosol filters can be divided in two categories, fibrous filters and membrane filters.
Fibrous filters are made of organic (e.g., cellulose) or inorganic (e.g., glass or quartz) fibers in a
randomly oriented loosely bound arrangement. Membrane filters have a much more regular and
smooth surface as they are made of a thin layer of a polymer (e.g., cellulose ester or Teflon) with
fine capillary pores. The Nuclepore filter is a special membrane filter with uniform structure and
pore size distribution.
The basic filter sampling train consists of a filter holder assembly, a pump and a volume measuring
device. The technique is easy and straightforward and can be applied with simple equipment that is
available in almost every chemical laboratory. Furthermore, filter sampling is still prescribed in
most official standard methods. Two types of filter units may be recognized, the high volume
samplers and the low volume samplers. An advantage of high flow rate sampling is the large filter
surface exposed to the air stream that after the end of sampling can be divided into several smaller
parts for analysis by different analytical techniques. Besides, it is the standard method for
collecting particulate material. Low flow rate filter holders can be designed to adapt to the specific
input sample requirements of XRF and for a clear definition of particle size range collected. When
applying high volume sampling several hundred m3 of air and thus up to 1 g of dust is collected (on
a large filter surface). Low volume samplers collect only a few mg of air particulates on a circular
filter surface of usually 27 or 47 mm diameter.
Particulate material is often leached out from the filter material before analysis in for instance
organic analysis of polyaromatic hydrocarbons (PAH) and inorganic analysis of a number of
constituents by atomic absorption spectrometry. In XRF the analysis is almost exclusively
performed directly from the aerosol coated filter. This avoids a time consuming and contamination-
prone sample pretreatment but imposes a strict requirement on the filter material used: it should be
pure so as not to affect the results of multi-element analyses with high and variable blanks.
In order to be useful for XRF, filter materials also need to fulfill a number of requirements of
physical nature:
• A high filtration efficiency, function of particle size and air velocity;
• A low pressure drop across the filter and reduction of the flow rate by dust loading;
• Weighable without large uncertainties from moisture absorption;
• Non-reactive with gaseous pollutants in the atmosphere;

55
 • Suitable secondary characteristics as hygroscopic, tensile strength, ease of handling,
physical stability and non-electrostatic properties;
• Collection of particles primarily at the filter surface, so as to reduce or simplify absorption
corrections for the exciting and fluorescence X-rays.
Of prime importance for XRF analysis is that the filter is sufficiently thin and uniform to keep the
background in the X-ray spectrum to a minimum.
Information on the physical properties and the filter efficiencies of many commonly used filter
materials can be found in manufacturer’s documentation11 and in textbooks and research papers20-21.
The physical properties of glass or quartz fiber filters are excellent but they lack the purity required
of a support material for inorganic analysis.
Impurity levels of 24 different elements were measured by instrumental neutron activation
analysis22. It is a safe precaution, however, to analyze blank values regularly in order to control the
blank levels of the specific batches used (blank values may vary strongly from batch to batch).
Special aerosol filters for chemical analysis are obtainable at a slightly higher price.
The materials used for analysis include cellulose fiber filters (e.g.Whatman numbers 1,40,41,541)
and filters based on Microsorban polystyrene, glass fiber, cellulose in membrane, Teflon
membrane, and Nuclepore. On the basis of a number of requirements including purity,
homogeneity, tensile strength, cost and ease of handling, cellulose fiber filters are often used for
XRF and other multi-element methods such as Instrumental Neutron Activation Analysis.
Alternative choices (especially as 47 and 25 mm diameter filters for low volume sampling) such as
Nuclepore polycarbonate membranes and cellulose ester membrane filters are often used. Particle
retention efficiency for this latter material is excellent but the high flow resistance reduces the
attainable flow rates. An important difference between the fiber cellulose filter and the cellulose
and polycarbonate membrane filters is connected with the penetration of the particulate matter
within the filter substrate.
Membrane filters can be approximated as screen filters which collected most of the material at or
very near the surface. Cellulose fiber filters are typical depth filters in which the particulate matter
penetrates to some extent, thus giving rise to radiation absorption in the filter23.

5.- Liquid Samples

Not always is possible to make the quantitative determination on the samples directly, often is
necessary to apply some chemical procedures to transform these samples to other state in order to
make the analysis feasible. This happens in the cases in that the sample (e.g., of metallurgical
origin), appears in variety of forms; or when the inhomogeneity and the effects of matrix are severe
(e.g., in minerals); or when the standard internal addition is necessary; and most of case, in no
routine analysis there are no reference standard available. In such cases, standards and samples can
be taken to liquid solutions.
The analyst who makes that decision must consider the advantages and disadvantages that present
the manipulation of liquid samples.
Advantages
1. Homogeneity. One of the main advantages is the homogeneity that is obtained in liquid phase.
It is admitted a highly stable suspension in which the magnitude of the amount of solute does
not exceed the colloidal state. In this case this concentration of solute had to be so small that a
possible attenuation of the analytical emission would be imperceptible.
2. The dilution reduces matrix effect. It just have been mentioned the advantage of the treatment
by dilution to reduce the matrix effects. The dilution is applicable if the intensity of the analyte is
not affected providing a good statistic of count. It is necessary to consider that the intensity of the
analyte not decrease in the same degree as the concentration of the element24-25.

56
Table 5 shows that the concentration of Zr decreases 250 times while the measured intensities
decrease approximately 10 times less.

Dilution Concentration mg/ml ZrKα

--- 40 20570
1:2 20 17163
1:10 4 7098
1:50 0,8 2161
1:100 0.4 1358
1:250 0.16 881

Table 5. Effect of the dilution on the intensity of ZrKα in liquid acid matrix

3. Preparation of standards. Synthetic standards of calibration are prepared easily, as well as the
aggregate and homogenization of internal standards. Calibration curves with solutions of the pure
elements can be prepared not being needed standards of similar composition to the samples.
4. Samples of different forms can be analyzed.
5. Neither surface nor heterogeneity effects
6. It can be preconcentrated. If the analyte is in low concentration a suitable treatment to
preconcentrate it can be made evaporating the solvent by heating, reducing the volume or until
dryness. Others more complicated preconcentration methods include precipitation of the analytes,
ion-exchange techniques, solvent extraction, ashing.
7. Can be made thin films. This is one of the most successful techniques, and all preconcentration
methods will be seen in the next section.
Disadvantages
There are some limitations:
a - The sample is destroyed (if that is not its original state).
b - High dilutions decrease sensitivity. Dilution of sample beside of decreasing the amount analyte
becomes matrix lighter and this matrix produces a higher background radiation.
c - Low fluorescence intensity of light elements.
d - High background by greater dispersion.
e - Heating and gaseous evolution during irradiation. Primary radiation emitted by an x-rays tube,
causes heating of the exposed solution; the solvent can volatilize, fact that must be worse in organic
volatile solvents. Radiolysis with formation of bubbles, and in some cases precipitations of the
specimen can take place. These disadvantages can be avoided or at least reduced, by cooling the
specimen; working at low power of excitation or decreasing the time of irradiation. This is feasible
if the intensity of measured fluorescence is not reduced to values that affecting the statistical
precision. To avoid bubbles solutions with water previously boiled and acid previously warmed up
may be prepared, and finally it must not use peroxides.
In the case of radiolysis, precipitation can be avoided with the use of complex agents such as
tartaric, citric, or oxalic acids.
Effect of Acids. The nature and concentration of acid used in the preparation of solutions have an
influence that can be considered: the more absorption coefficient of acid the lower intensity of the

57
measured line and this is accentuated in the determination of light elements1. The magnitude of this
influence depend of the atomic number and concentration of the acid, the most common used are:
- Fluorhydric acid
- Nitric acid
- Phosphoric acid
- Sulfuric acid
- Perchloric acid
- Chlorhydric acid
Precautions. Corrosive liquids, such as hydrofluoric acid, damage instruments and could break the
supported membrane. The infinite thickness is much greater for liquids than for solids and
considerably varies with the wavelength and the concentration of the analyte in the matrix.
Liquid Cell Samples. Cell samples provided by the manufacturers have different forms, the most
common are made from aluminum, titanium, and araldite that is resistant to acids. Disposable cell
that fit all commercial instruments are dealed with Spex Ind. In general cell bodies are made of
plastic (Lucite, polyethylene, polypropylene, nylon and Teflon) with a ring prepared to work in
vacuum,
Support Films. The most commonly used is polyethylene tereftalate or Mylar. It is presented in
several thicknesses according to the wavelength that will be measured. The thickness 3 µm is used
for light elements, but is most fragile.
In general Mylar presents inclusions of high absorption, like Sb that is used in the manufacture of
it. The total concentration of impurities is relatively small, ~ 0.2% in weight in ashes. But the
average of atomic numbers (z) in the inclusions is high and anomalies are produced when elements
of λ > 6 Å are measured.
Polypropylene is very recommendable by the low of impurities it contained (<0.005 % ash). The
average of Z is smaller to half respect to Mylar and its coefficient of absorption is smaller in a
factor ~ 5. The thickness is of 12 mm. "Kampton" is a film of 7.5 mm and is free of calcium, but it
is of high cost.
Supported Samples. Commonly used support include the following: filter paper (Whatman 41 is
most recommendable), chromatographic paper, Fiberglas or other glass-fiber, thin Mylar,
polyethylene film, Millipore filter, Nuclepore filters, ultrathin collodion or Formvar film, cellulose
powder, activated carbon, ion-exchange resin-impregnated paper, ion-exchange resin-membrane,
ion-exchange resin granules, microscopy-slide cover glasses, etc.
Samples Derived from Solutions. The simplest technique to obtain thin films consists of
depositing small volumes of solution, using micropipette with disposable tips to avoid
contaminations on the support, and evaporating to dryness. If filter paper is used as supported the
area can be delimited previously with an imprinted wax ring26. This can be made simply,
introducing metallic aluminum ring in melted wax and imprinting on the paper for few seconds
until the solidification of wax. On that area is deposited between 1 and 1.5 milliliter of sample.
The limits of detection of elements deposited in filter paper are 10-2 to 10-5 % (100 to 1 µg/g).
Working with colored solutions has verified that the most uniform distribution is obtained with
paper Whatman; however, still in this one, an intensity gradient has been observed from the center
of the deposited area to the periphery (~10 % more). If the drying is made in high temperature the
effect is increased. The chromatographic effects have a great influence in the reproducibility. The
factors more important are: the temperature of the solution, the dilution, and the type of filter. The
distribution is less uniform when increase the temperature and the pore size of the membrane. The
best reproducibility is obtained with solutions at room temperature and very fine pore membranes.

58
 A successfully method is to impregnate cellulose powder chromatographic quality (1 or 2 g) with a
small volume of solution (1 to 1.5 milliliter by gram of cellulose). It is placed in oven to 80°C
during several hours, after complete dryness is homogenized by mixing in a mixer mill and pressed
directly obtaining an excellent pellet. The technique allows the aggregate of internal standards as
well as to make standard additions of some element. This method was applied with good results in
the analysis of minerals and rocks, dissolving by fusion and acid treatment and the determination of
trace elements in waters of lakes and rivers27.

5.- Preconcentration
The determination limit for elements of atomic number Z > 25, in liquid is usually between 10-3
10-4 % whereas for light elements in the order of 10-2 %. Analysis near the detection limit leads to
an increase of the relative standard deviations (S % from 20 to 50. Basic technique to decrease the
detection limits consists of preconcentrating the analyte before the determination. Usually, small
amount of the liquid sample is supported on some type of substrate. There are a great amount of
supports that can be used as substrates. The technique is applied for determination of vestiges of
elements.
Liquid samples can measure directly placing some milliliters of solution in a cell sample. In
favorable cases WDXRF, can detect concentrations down to 1 µg.mL-1. Preconcentration
techniques have to be applied in the case of low concentration of analytes (trace elements).
IUPAC defines preconcentration: the operation (or process) by which the relation between the
amounts of microcomponents (traces) and macrocomponents (matrix)"increases". This process
improves the detection limits, reduces matrix effects, facilitates the calibration, but the time of
analysis is increased, with risks of contamination or losses of trace elements.
With respect to the preconcentration by physical procedures, evaporation and dry film deposits,
have already been discussed.

5.1.- Ionic Exchanges

Ion exchanges are a reversible exchange of ions between the resin and a liquid, resulting no
changes in the resin.
The following equations represent the ion-exchange reactions:
Cation-exchange resin:
RH + C+ RC + H+

Anion-exchange resin:
ROH + C- RC + OH-

They can be used in batch placed directly in the solution, or column. The resins commonly used are
very selective; the abundance of alkaline ions in waters competes with transition metals in its
fixation. This must be considered in its application. If the amount of alkaline and earth-alkaline is
limited, cationic resins can be used. Is important to work particularly with resins with size of
granule is < 50 6,3 mm. The general procedure consists: the solution is allowed to stand a certain
time or is shaken with an amount of resin granules or liquid, or circle of membrane or resin-paper
impregnated paper. The granules are separated by filtration, dried at room temperature and to put
under irradiation the specimen directly or pressing previously to obtain a pellet. The liquid resin are
separated by decantation and used in a liquid cell. Membranes or papers are rinsed, dried and
mounted in the cell.

59
Special cares must be taken not to saturate the resin. In dilute analyte solutions, it may be necessary
to purify the resin by washing with pure eluent.
Wide type of resins is available in the commerce. It is necessary to make notice certain It is
important to show some disadvantages working with resins: sensitivity is not optimal because the
preconcentration factor (relation between the weight of original sample to final weight) is around
5000 (example 100 mg of resin by 500 milliliter of water); It could be necessary to add a binder to
do a pellet; absorption and particle effects can be significant in direct measurements on the resin.
Working with membrane or resin –impregnated paper reduces the previous problems.

5.2.- Membranes Impregnated in Resins

They are excellent means of preconcentration with quantitative recovery in the region of ppb.
Include resins of ionic exchange finely divided in a polymeric structure. They are very thin: 5
mg.cm-2 and provide a matrix with low atomic number, can be exposed directly to the radiation
after the exchange has taken place. This is simply made by filtration of the solution sample through
the membrane, or immersion producing an intimate contact between the solution and the fraction of
impregnate membrane.
An excellent work on applications of cationic and anionic membranes28 used cationic SA-2, has
sulfonic acid functional groups containing ~ 50 % of resin IR-120 and 50% of cellulose. The
filtrate technique consists of passing the solution sample very slowly (~1,5ml.min-1) in a filtration
apparatus that contains the membrane (Figure10). The filtrate is passed 6-10 times again through
the same filter to assure the fixation. In this case was used membranes SA-2 in rainwater analysis
very successful, because these have a low content of alkaline and earth-alkaline, and SA-2 has a
significant affinity. Chelex-100 (Bio Rad Laboratories) that is efficient in collecting trace metals.
The enrichment factor is 1250, and 1 ppb can be detected with a 10-15 % of precision. A work
where the action of two resins was combined is the one of Kingston and Pella that determined Nor,
Mn, Cu, Zn and Pb in sea water, at a 2-4 level mg.L-1 with 0.2 mg. L-1 standard deviation. They
used a first column with Chelex-100 separating Na, K, Ca and Mg. After evaporating the filtrate
and eliminating ammonium salts by subliming, passed the solution through filter SA-2, fixing the
mentioned cations.
Among other existing membranes they are:
1, P-81 de Whatman
2, WA-2 of Revé Angel. (Acid weak).
Resins used for preconcentration must have some characteristics, particularly in the water analysis;
1. No affinity for alkaline and earth-alkaline
2. High stability constant for heavy metallic ions
3. Stable molecular structures
4. Easy immobilization on a substrate
Theoretical and practical considerations indicate that the membrane 2-2'-diamino-diethylamine,
called diethylamine (DEN) satisfies better these requirements. Smits and Van Grieken29-31ed in
detail DEN, they found recovers between 90 and 100 % of Cr 3+, Fe3+, Co2+, Ni2+, Zr+, Ag+, Cd+,
Eu3+, Hg2+, Pb2+ and UO22+. Practical detection limits about 0.5 mg.L-1 and even smaller, with
exactitude and precision of 10% can be reached.
It is interesting the system automatic of preconcentration and analysis EDXRS, called SCPM
(Sample Collection and Preparation Module) and developed by Ho and Lin32 they put in a tank a
roll of impregnated paper of SA-2 or SB-2, an aliquot of 500 milliliter of water is used. The used
part is cut and carry directly to the measurement equipment (EDXRS) by automatically

60
transporting. A precision from 1 to 2 % for 10 mgL-1 and limits of detection of ~ 1mg.L-1 were
reported.

Figure 10. Filtration equipment and membranes

5.3.- Preconcentration by Precipitation and Co-Precipitation

It is very attractive means to use with XRS, by its simplicity and efficiency by the amount of
organic reagents available for the precipitation. A selective precipitation is not essential. The
simplest procedure in waters or other solutions is the precipitation with organic reagents and direct
measurement of the filtered. Between the most used reagents one of it is the dithiocarbamate
(DDTC) that forms metal-chelates water insoluble. Luke33 established the conditions for the
determination of trace metals by precipitation with NaDDTC.
Ammonium pyrrolidine-dithiocarbamate (APDC) is the most recommendable when the
concentrations are very low.
1-(2-pyridylazo)-2 naphtol (BREAD), is a very soluble chelating agent in hot water and alcohol,
and forms insoluble chelates in cold water. The enrichment factor of 105 can be obtained.
Hydrated iron oxidize at pH 9 precipitates Ni2+, Cu+2, Zn2+, Pb+ quantitatively in seawater.
In all procedures described must be used high purity reactives to avoid contaminations that could
alter the analytical results.
Extraction by solvent
It is not used commonly in XRS because the preconcentration coefficients are not very high. If it is
necessary to evaporate, this requires time. The enrichment factor is between 10 and 50. As
example, it can mention the preconcentration of microamounts of U, by extraction solvents and
their direct determination in organic phase by XRS. 200 mL of uranium solution in chloridric or
nitric medium is contacted with 10 mL of solution of Tenoyltrifluoroacetone (TTA) in benzene or
chloroform; or with 10 mL of Trioctylamina (TOA) if the uranium is in medium. The enrichment
factor is 1:20. The organic phase is placed directly in the spectrometer, determining inferior limit
to 0.12 µg.L-1. Another selective extractor for uranium is 3-n-octylphosphine34 used in solutions
from plants.
Other reagents used are:
APDC in chloroform
DDTC in carbon tetrachloride
Immobilization on activated carbon: It is good adsorbent of organic and colloidal material. The free
ions are not adsorbed, but when adding a chelating agent becomes absorbable form. For example 8-

61
quinolinol is a good chelate agent, its chelates have high stability constant for many ions, but not
for earth-alkalines. Vanderborght and Van Grieken35 working with 8-quinolinol recover 20 ions
quantitatively with an enrichment factor ~10000. Reproducibility of 5% with linear answer in
EDXRS up to 1 mg.L-1 of metal concentration could be reached.

6.- Ashing Techniques

Solid organic samples may be preconcentrated by ashing, for concentration of the analyte from
0.01 to 19 µg.g-1.It can distinguish four distinct processes:
• Dry ashing: only for combustible samples which are ignited to a carbon form in a muffle,
and collected for analysis. The process is not recommended for volatile
analytes.
• Wet ashing: samples are treated with a hot mixture of acids generally sulfuric, as
dehydrating; and nitric or perchloric, as oxidizing. The heating with the acids
is repeated until a clean ash is obtained. No loosing of analytes but very
time-consuming is attainable with this process.
• Dehydration-ignition: ashing is obtained by heating with a dehydration acid (sulfuric). The
residue is ignited in a muffle and carbon becomes a clean ash. This process
produces no loss of volatile analytes and is much faster than wet ashing.
• Wet ashing and combustion: for combustible samples only. Samples are heated with
benzenesulfonic acid and burning as in dry ashing.
In all the case the ash may be placed on a Mylar, a filter paper, a glass-fiber or Millipore filter and
analyze directly in the equipment.

7.-Organic Liquid Samples

Petroleum samples and their derivatives, light and heavy hydrocarbons, lubricating oils, etc are
analyzed. Different elements mainly Ca, Mg, Ba, V, Zn, Ni, P, S, Pb are determined.
The concentrations are between 0.05 and 2 % thus, as well as traces. Also the additives that are
added to modify the physical properties are analyzed. It must work with external standards, or adds
an internal standard miscible with the matrix of the sample to compensate variations in absorption
coefficients of the matrix. The concentration of S in hydrocarbons in different samples alters the
absorption coefficients. This leads to make corrections by its presence.
It is recommendable the use of polypropylene as a window in the cell samples, particularly in light
trace elements analysis.
A very interesting application in lubricating oils is the determination of metals produced by the
wearing on the engines. The concentration of metals shows an abnormal behavior of the engine.
The oil is filtered by using Millipore (0.8 µm) and the particles are analyzed on the filter and on the
filtrate36.
Total-Reflection X-Ray Fluorescence Analysis
TXRF is a variation of energy-dispersive XRF with one significant difference. In TXRF the
primary beam strikes the sample at a glancing angle of less than 0.10. Owing to this grazing
incidence the primary beam is totally reflected.
TXRF is primarily used for chemical micro and trace analysis. Small quantities of solutions or
suspensions are placed on optical flats, e.g., quartz glass, serving as sample supports. After the
evaporation, the residue is excited under the total reflection conditions and the characteristic
radiation from the elements that constitute the sample is recorded by a Si(Li) detector as an energy-

62
dispersive spectrum. The spectral background is eliminated due to the high reflectivity of the
sample support.
This mode of XRF does not permit the nondestructive analysis of the sample, but it can be analyzed
trace element in a sample.
Because of TXRF is a method of microanalysis, it is directly applicable when only a small sample
amount is available or when only a small part of larger amount, specimen, can be taken. In order to
get a representative result by mean of a specimen, the sample has to be homogeneous or
homogenized carefully before the analysis. For the analysis only suprapure reagents may be used,
acids be finally purified by sub boiling and water should be prepared by a double-stage
deionization or bidistillation.
Highly pure acids shows residual impurities, especially elements like Mg, Al, Fe, Cu, Zn and Pb
may show high blank values when concentration in order of ng. mL-1 has to be analyzed.
Purification of by these liquids and even the bidistilled water by sub boiling distillation has to be
done. Heating below the boiling point vaporizes the liquid. The vapor is condensed at a cooling
finger, and the purified condensate is collected in a small flask. Impurities are significantly below 1
ng. mL-1.
For the preparation of samples, only vessels made of quartz, PTFE (polytetrafluorethylenes, as
Teflon), or PP (polypropylenes) should be used.
The specimen should be placed on highly clean sample support (carriers). One procedure of
cleaning carriers consists in placing the carriers in a solution of Extran diluted in bidistillated water
during 48 hs. After that thoroughly rinsing with sub boiling distillated water. Put into in a 10 %
nitric solution (all reactive prepared by sub boiling distillation) during 48 hs. Finally drying at
putting into clean Petri dishes, covered by the top, they kept inside until needed for analysis. The
cleaning of the carrier is checked (before the specimen deposition) by recording of the spectrum of
each carrier within 100 s. The carriers, whose spectrum show impurities, should be sorted out and
cleaning again.
Preparation of Samples. When the trace analysis is to be performed in a small specimen
representative of a larger amount of sample, first of all it must be homogenized, and the sample
matrix should preferably be separated.
The operations are more difficult for solids than liquids and more difficult for inorganic than
organic or biomaterials.
A droplet of 10 µL is pipetted on a center of circular carrier. Pipettes of 1-10 µL are normally used,
the polypropylene tips have to be used only once. It is recommendable to put the carrier on a
marked center pattern to help the best centered of the droplets (Figure 11). The droplets are dried
by evaporation. For that purpose, the carriers are placed on a hot plate or under an infrared lamp.
The residue may be seen as a spot of about 1-5 mm or may be hardly visible. In the case of organic
solvents they can be dried up with the help of a small PTFE cylinder pressed against the carrier37.

63
 Solid Liquid

An small part of the An aliquot

large amount of sample

Digestion for organic, or matrix

Dissolution, suspension, decom- removal and trace concentration
position, or digestion

An addition of an
internal standard

Placed on the carrier and dried

TXRF Measured

(a) (b) (c)

Figure 11. (a) Sample deposition on the reflector; (b) Drying with an infrared lamp; (c) a pattern to
help in centering the sample on the reflector

Preconcentration by Hydride Generation

Metals having the properties of reducing to hydrides could be separated from the sample. By using
sodium borohydride in acid medium some

64
References
1 E. Bertin, Principles and Practice of X-Ray Spectrometric Analysis. New York, 1975, pp.
701-806.
2 Bennet, Harry; Oliver, Graham J. XRF Analysis of Ceramics, Minerals and Allied
Materials. John Wiley & Sons, 1992.
3 Birks, L.S. X-Ray Spectrochemical Analysis. Second Edition, Interscience Publishers, New
York, 1969.
4 Burke, Victor E.; Jenkins. Ron; Smith, Deane K. (Eds.). A Practical Guide for the
Preparation of Specimens for X-ray Fluorescence and X-ray Diffraction Analysis. Wiley-
VCH, 1998.
5 Jenkins, Ron. An Introduction to X-Ray Spectrometry. Heyden, London - New York -
Rheine, 1980.
6 Jenkins, Ron. X-ray Fluorescence Spectrometry. Second Edition, John Wiley & Sons, Inc.
1999.
7 Jenkins, Ron; De Vries, Johan Louis. Practical X-Ray Spectrometry. MacMillan, London,
1976.
8 Van Grieken, Rene E.; Markowicz, Andrzej A. (Eds.). Handbook of X-ray Spectrometry:
Methods and Techniques. Second Edition, Marcel Dekker Inc., New York, 2002.
9 R. Tertian and F. Claisse, Principles of Quantitative X-ray Fluorescence Analysis, Heyden,
London, 1982, pp. 317-333.
10 Bhuehler Ltd. AB Metal Digest. Materials of polishing.
11 H. L. Baker, Advances in X-ray Anal., 2001, Vol. 44 pp. 398-404.
12 F. Feret and R. Jenkins, in A Practical Guide for the Preparation of Specimens for x-ray
Fluorescence and X-ray Diffraction Analysis, ed. V. E. Buhrke, R. Jenkins and D. K. Smith,
Wiley, New York, 1998, pp. 35-122.
13 K. Matsumoto And K. Fuwa, Anal. Chem., 1979, 51, pp. 2385.

65
14 M. Guevara and S. P. Verma, X-Ray Spectrom., 1987, 16, pp. 2355.
15 K. Norrish and G. M. Thompson, X-Ray Spectrom., 1990, 19, pp. 67.
16 Y. N. Hua and C. T. Yap, Anal. Sci., 1994, 10, pp. 867.
17 H. M. West, J. Cawley and R. Wills, Analyst, 1995, 120, pp. 1267.
18 Maggi Loubser , Christien Strydom, Herman Potgieter, X-ray Spectrometry, 33(3), pp.
212
19 Giauque, R. D. and col. X-ray Fluorescence Analysis of Environmental Samples, ed.
Dzubay, pp. 153.
20 R. K. Skogerboe, D. L. Dick, Lamothe, P. J. Atmos. Environ.
21 R. G. Stafford and H. J. Ettinger, Am. Ind. Hyg Assoc J. 1971. 32(5), pp.319-26
22 R. Dams, K. A. Rahn and J. W. Winchester, Environ. Sci. Technol., 1972, 6, pp. 441.
23 F. C. Adams and RE Van Grieken, Anal. Chem., 1975, 47(11), pp. 1767.
24 D. V. Leyt, CNEA (Argentina) Inf. PQ/QA/56, 1978, V II, pp. 117.
25 T. J. Cullen, Pittsburgh Conf. Anal. Chem. Appl. Spectry, 1963.
26 J. A. Smith and RE Van Grieken, Anal. Chim. Acta, 1977, 88, pp. 97
27 H. J Rose and F. Cuttita, Adv. In X-Ray Analysis, 1967, 11,23.
28 RE. Van Grieken, Anal. Chim. Acta, 1982, 142, 3.
29 J. A. Smits and RE. Van Grieken, Anal. Chem. 1980, 52, pp. 1479
30 J. A. Smits and RE. Van Grieken, Anal. Chim. Acta, 1981, 123, 9
31 J. A. Smits and RE. Van Grieken, >Int. J. Environ. Anal. Chem., 1981, 9, 81.
32 J. S. Y. Ho and P. C. L. Lin, Int. Lab. >July/Aug., 1982, 44
33 C. L. Luke, Anal. Chim. Acta, 1968, 41, pp. 237
34 N, Barris, M. Zlozilo, X-Ray Spectrom., 1978, 7, pp. 31
35 Vanderborght, B. M. And R. Van Grieken, Anal. Chem. (1977), 49, pp. 11.
36 G. R., Humphrey, Joint Oil Analysis Program Technical Support Center, 1996, JOAP-TSC-
TR-96-02.
37 A. Prange, A. Knöchel and W. Michaelis, (1985). Anal. Chim. Acta 172.79.

Graciela Custo, Dora V. de Leyt and Cristina Vázquez

Unidad de Actividad Química
Comisión Nacional de Energía Atómica
Av. Gral. Paz 1499 B1650KNA. Buenos Aires. Argentina
custo@cnea.gov.ar

66
 3
Chemometrics in Atomic Spectroscopy

Alejandro C. Olivieri

Chemometrics
The term chemometrics was coined only three decades ago, when the swedish professor Wold used the
word chemometrics to describe his area of expertise in a form to apply for a research grand. This are
was the development of partial least-squares regression, currently one of the most popular techniques
for multivariate calibration. The beginning of chemometrics, even without this formal label, can be
traced to the very first applications of mathematical methods to chemical problems. Today, in a broad
sense, the discipline groups data processing tools as simple as univariate linear regression, and as
complex as tensorial analysis or artificial neural networks. It is not only a research field in itself,
having prestigious publications such as Journal of Chemometrics, Chemometrics and Intelligent
Laboratory Systems, and Journal of Chemical Information and Computer Sciences, edited by Wiley,
Elsevier and ACS respectively, but also provides support to other disciplines as diverse as psychiatrics
and analytical chemistry. In the latter case, chemometrics significantly contributes to the resolution of
analytical problems of growing difficulty. A paradigmatic example is the quantitative prediction of a
component in complex mixtures, starting from instrumental signals which are intrinsically unselective:
chemometrics is a non traditional approach to this type of problems, which tries to replace the classical
resource of chemical derivatization or search of instruments with higher resolution. The growing
concern for the consumption of safe feedstuff, the environmental pollution and fair sport – to mention
just a few cases – are sources of analytical problems for which the traditional concept may not be
practical in terms of time and/or cost.
The purpose of this section is to discuss some topics which are relevant to current chemometrics,
which are related to atomic spectrometry. Some selected chemometrics tools (the list is not
comprehensive) are:
• Principal component analysis

67
• Uni- and multivariate calibration
• Aritifical neural networks
• Surface response optimization
• Genetic algorithms
These techniques are regularly employed to reach certain chemometric aims, such as the following:
• Classification
• Quantitation
• Experimental design and optimization
• Variable selection
• Regression and modelling

Classification
We will briefly discuss how a classification of objects can be made using chemometric tools. A tipical
case involves the study of a set of samples, knowing a certain number of properties for each of them.
One can assign numerical values to these properties, for example, the concentration of a series of
metallic elements, determined by atomic spectrometry. The processing of these data can be done using
different techniques: supervised and non supervised. Supervised techniques require the previous
knowledge of objects belonging to known classes, whereas non supervised tools do not require a
previously known classification. Examples of supervised techniques are linear discriminant analysis
(LDA), k-nearest neighbors, SIMCA (soft independent modelling class analysis), and artificial neural
networks (ANN). On the other hand, non supervised techniques usually employed are principal
component analysis (PCA) and clustering.
The most popular non supervised tool, employed in many literature works on atomic spectrometry, is
principal component analysis or PCA. Assume we know instrumental data or elemental concentrations
for a set of I calibration or training samples. If J different data for each sample are known, we can
build a matrix of size J×I containing these data (Figure 1). PCA analysis intends to reduce or compress
the analytical information contained in this matrix, yielding, through a mathematical transformation, to
a matrix of lower size, known as the scores matrix. If PCA succeeds, usually a reduced number of
rows of the scores matrix (Figure 1) are significant, in the sense that they will explain the largest
portion of the variation contained in the original data matrix. The remaining scores are non-significant.

Significant scores

68
Matrix of training Non-
data (J x I ) significant
After this first compression step or data reduction step is accomplished, the original values are
transformed into significant scores, selecting, for each available sample, the significant scores. This is
done in order to represent the position of each sample in an n-dimensional diagram. When the largest
portion of the variation contained in the original data matrix is represented by the first two scores, each
sample or object will be represented by these two values: the first two scores (Figure 2). These
numbers can be used to locate the position of a sample in the bidimensional space of the first two
scores (Figure 3).

Sample i Score ti1

Score ti2

First two significant

scoressignificativos
((2 x I )

Non-significant
socres

Matrix of scores (I x I )
Figure 2. Correspondence between samples and the first two scores

69
 Sample i
Score ti2

Score ti1
Figure 3. Location of a sample using the first two scores in a
bidimensional plot

If this analysis is correct, and the first two scores allow one to locate samples in a bidimensional plot,
and if these scores are enough to differentiate objects in some way, the complete plot for a set of
samples will allow to distinguish separated regions grouping similar objects. Figure 4 illustrates a
successful separation between two object classes, A and B, using PCA with the first two scores.
Second score

Samples of
type A Samples of type B

First score
Figure 4. Separation of samples of two types using PCA with two scores

Mathematically, the compression of the information contained in the data matrix is the critical step
for PCA. A technique very employed in chemometrics for the efficient data compression is the
projection onto the space of the eigenvectors of the matrix (XXT). Several algorithms exist for

70
obtaining these eigenvectors; a very efficient one is based on a matrix decomposition known as
singular value decomposition which consists in decomposing the matrix X (size J×I) as the product of
three other matrices:
X = U W VT (1)
The matrices U (J×I), W (I×I) and V (I×I) satisfy the following conditions:
• The columns of U arte orthogonal, in such a way that UT×U = I, as the columns of V, in such as
way that VT×V = I (I represents a unit matrix).
• The matrix W is diagonal and its diagonal elements are non-negative (the off-diagonal elements
are all zero).
The diagonal elements of W are called singular values of X. Mathematically, they are the square roots
of the non-negative eigenvalues of (XXT); from a chemical point of view the measure the contribution
to the variation which can be explained by each of the principal components of X.
The columns of U are the eigenvectors of (XXT), while the columns of V are the eigenvectors of
(XTX). The columns of U are usually called loadings or factors, or latent variables, in contrast to the
manifest variables, which are experimentally accessible (the latent ones have to be found by
mathematical operations).
Joining the product W VT into a single matrix T, the decomposition equation (1) can be written as:
X=UT (2)
which is the base of PAC, where T is the matrix of scores. Figure 5 illustrates this operation in a
graphical way.

U
X Matrix of T
Training matrix eigenvectors Matrix of
(J x I) of XXT scores (I x I )
(J x I )

X=UxT
Figure 5. Decomposition of a data matrix in principal components

The method of singular value decomposition is usually employed to identify variation sources. A
variation source is any phenomenon able to produce a variation in spectra (or concentration) from
sample to sample. There are several ways of estimating the sources of variation: a popular one is to
consider the relative contribution of each principal component to the overall variance, calculated as:

71
 A
∑ (Wi ) 2
i =1
% Variance explained by the first A factors = 100 I
(3)
∑ (Wi ) 2
i =1

where Wi is each of the diagonal elements of the matrix W, and (Wi)2 is the corresponding eigenvalue
associated to the principal component i.
Since each principal component contributes with a decreasing portion of the total variance, a usual
criterion is to take the first principal components which, collectively, explain a certain percentage of
the overall variance. Figure 6 illustrates the typical behaviour of a set of principal components: while
the singular values decrease, their contribution to the overall variance also decreases. In the case of
Figure 6, for example, the first three principal components explain more than 99% of the spectral
variance, which leads to the conclusion that there are three sources of variation in X.

Explained variance

Figure 6. explained variance as a function of the principal component number

principal
99% of the
Variance

variance explained
Figure 6: explained variance as a function of the principal component number.

Principal component
Figure 6. Explained variance as a function of the principal component number

The analysis of the number of variation sources is extremely important. Suppose that this study
indicates the a number A of factors which explain a significant percentage of the variance is lower than
the number of training mixtures I. Since the first A factors are enough to explain practically all the
spectral behaviour of matrix X, it is not necessary to use the complete matrix U in equation (2), but the
columns from A+1 to I can be removed, leaving a matrix formed by only the first A eigenvectors,
called UA (size J×A). The remaining eigenvectors can be discarded because we consider that they only
model spectral noise.
In this way, the matrix X can be rebuilt with a simplified equation:

72
 X = UA TA (4)
In the latter equation, we called TA to the matrix of scores estimated with A factors. This new matrix
TA, although having a size considerably lower than the original data matrix, plays however a similar
role, since the relevant information present in X has been compressed in an efficient way.
The process of compression can be illustrated with the series of images in Figure 7, which show the
photograph of a flower, considered as a matrix of points, which can be compressed using principal
components, and then "rebuilt" using equation (4) written in terms of the selected A components. The
image corresponding to A = 1 was rebuilt using only the first principal component, which is the one
contribution with the largest portion of the matrix variance. Using more and more principal
components, the image becomes more clear. However, it can be seen that with a few factors, the
relevant information if retained by the compressed data matrix.

Total image

A=1 A=2 A=4 A=8 A = 16 A = 32

Figure 7. An image (top centre), rebuilt using a growing number of principal components
(bottom)

Concentrations (%) of elements in coloured glasses.

73
Classification examples using atomic spectrometry are now discussed. In the work entitled "Energy-
dispersive x-ray fluorescence analysis of modern coloured glasses from Marinha Grande (Portugal)",
whose authors are P. Valério, A. Markowicz, P. Kregsamer, M. F. Araújo, A. Pires de Matos, E.
Chinea-Cano and C. Carvalho, published in X-Ray Spectrometry 32 (2003) 396-401, coloured glasses
are classified using the elemental composition calculated by X rays. The following table contains the
values measured for a series of elements in glasses of different colour.
Figure 8 shows the analysis of principal components, taken from the above publication. The result is
that some samples are correctly classified according to composition and colour, but other appear
overlapped. This is due to the fact that the variation of the data matrix is not well represented by the
first two scores, and more principal components are required to correctly classify the objects. In the
above paper other combination of scores were tried, for example the fifth vs. the fourth. Still, the
classification using PCA was not completely successful.

Figure 8. Bidimensional plot of scores of the coloured glasses, after PCA.

Another technique employed for classification is artificial neural networks. These are algorithms
which require a previous training using objects belonging to known classes. Briefly, because the
mathematical details of the ANN are complex, they admit a set of input data, which are distributed
among neurons. Typically, a neuron exists for each input variable, for example, the concentration of
elements in a sample. These neurons form the input layer, and are connected with a second layer of
neurons, the hidden layer. The connection implies that the input data are transformed using a non-
linear mathematical function (usually of a sigmoidal nature) before passing to the hidden layer. Each
input neuron is connected to each of hidden neurons, and each hidden neuron receives information
from each input neuron, previously transformed by the transfer function. The hidden neurons receive
data in the form of linear combinations of the values transferred by the input layer. The coefficients of
this combination, or weights, are variables to be optimized by the algorithm. Finally, the hidden
neurons are connected to the output neurons, which form the output layer, with as many neurons as

74
output variables, for examples, de object classes. The connection between hidden and output neurons
is similar to that between input and hidden neurons.
The weights are adjusted using a procedure called training, in which the net is fed with known data
(element concentration and object classes). Once they are adjusted, they can be employed to predict
the class to which a new object belongs. These programs are more flexible than PCA, and can provide
intelligent responses in cases where the latter fails.
A literature example is the work entitled "Automatic Classification of Steels by Processing Energy-
Dispersive X-ray Spectra with Artificial Neural Networks", by J. F. Magallanes and C. Vazquez,
published in the Journal of Chemical Information and Computer Sciences, 38 (1998) 605-609. In this
work, ANNs are employed to classify steels, using training data consisting in % of metallic elements
determined by X rays in steels of known type. The following table reproduces the training data.

Once the neural network is trained with these data, the samples are placed in a bidimensional plot,
with the purpose of checking whether the classification has been successful. Figure 9 shows the
success reached by the program.

75
 Figure 9. Bidimensional representation of steels classified by neural networks.
The areas group similar members

Quantitation
The use of chemometric methods in quantitation problems allows one to improve the quality of
spectroscopic analysis in case of lack of selectivity. In this case a great variety of multivariate
calibration algorithms has been developed, and also artificial neural networks, with the purpose of
determining the concentration of an analyte in the presence of spectral interferfents.
In atomic spectrometry, spectral overlap is minimum, and for this reason there are only few works in
the literature using multivariate methods to improve the selectivity. One fo them is "Determination of
some rare earth elements by EDXRF and artificial neural networks", by F. Schimidt, L. Cornejo-
Ponce, M. I. M. S. Bueno, and R. J. Poppi, published in X-Ray Spectrometry 32 (2003) 423-427. In
this work, two different multivariate methods are compared: partial least squares regression (PLS) and

76
artificial neural networks (ANN), in order to quantitate praseodymium, neodymium and samarium,
whose spectra are partially overalpped (Figure 10).

Figure 10. Typical ECXRF spectra for Pr, Nd and Sm

When ANNs are employed to solve this type of problems, the output neurons carry information
regarding the concentration of a particular analyte. Once the net is trained with a set of samples of
known concentration, the final adjusted weights can be used to predict the concentration of the anayte
in a new sample.
The PLS multivariate calibration method, on the other hand, is closely related to principal component
regression (PCR), which will be described below. In PCR, an inverse calibration model is employed,
in which the concentration of the calibrated analyte in the calibration samples is known (yn, vector of
size I×1) and are linearly correlated with the scores contained in the compressed matrix of scores TA:
yn = TA vn + e (5)
where vn (size A×1) is the vector of regression coefficients, and e is a vector containing the model
errors.
The vector vn can be obtained from the latter equation,by pre-multiplying both sides by TAT:
TAT yn = TAT TA vn + e (6)
It is then necessary to multiply by the inverse of (TAT TA), that is, by (TAT TA)–1. This last operation is
trivial, since (TAT TA) is a diagonal matrix (because the columns of T are orthogonal), and the
inversion of a diagonal matrix is simply obtained by inverting each of the diagonal elements. Finally:
vn = (TAT TA)–1 TA yn (7)
T –1
Calling the matrix [(TA TA) TA] the pseudoinverse of TA and naming it TA+, equation (7) adopts its
final form:
vn = TA+ yn (8)

77
This last step completes the calibration. Obtaining the regression coefficients vn is analogous to the
process performed in univariate calibration, in which the first step one determines the slope of the
calibration graph.
In the prediction phase, the spectrum of a new sample is registered, and the signals are stored in a
vector x (size J×1). Before applying the prediction model it is necessary to project this vector onto the
space of the A factors of the matrix UA, since we cannot use the original data to estimate the
concentrations, "mixing" a real spectral vector with the compressed regression coefficients vn. The tA
(A×1) is obtained, corresponding to the new sample:
tA = UAT x (9)
This vector tA contains the sample scores, which act as "spectra" in the predictive phase of the model.
The latter repeats the inverse linear model:
yn = (vn)T tA (10)
From this last equation, the concentration of the analyte in the sample is estimated.
The method of partial least squares (PLS) intends to improve PCR introducing the values of the
calibration concentrations in the calculation of spectral factors. In this way, PLS employs
concentration-dependent factors, while PCR use factors which are independent on concentration.
In PLS two classes of spectral factors exist: weigth loading factors, contained in a matrix usually
called W, and loadings, contained in a matrix called P. The columns of W are orthogonal, while those
of P are not necessarily so, in contrast to PCR. It is important to notice that the columns of W are not
eigenvectors, but factors obtained by a technique different than PCR, whose elements depend on the
calibration concentrations of the analyte.
The obtainment of these factors is done by an iterative algorithm, in which the factors describing the
maximum possible correlation between the data matrix and the concentration vector of the analyte are
extracted one at a time.
Mathematically, PLS is summarized in the following elementary steps:
1) The data matrix X is projected onto the vector of concentrations yn, yielding the first weigth
loading factor, which is then normalized to unit length:
w1 = X yn / (ynT yn), followed by normalization.
2) The first score is obtained:
t1 = XT w1
3) The first regression coefficient v1 is obtained:
v1 = t1 yn / (t1T t1)
4) The first loading p1 is obtained:
p1 = XT t1 / (t1T t1)
5) Spectral and concentration residuals are estimated:
eXT = XT – t1 p1T
ey = yn – v1 t1
6) eX and ey are substituted by X and yn respectively in step 1) and the algorithm continues
until the desired number of factors A.
We now describe these steps in a qualitative way:
Step 1. In this step of the algorithm, it is assumed that we only know the concentrations of a single
component, in this case the analyte 1, in the calibration mixtures. A classical least squares (CLS)
analysis is applied, in this case assuming tha only analyte 1 is present in the calibration mixtures. In
other words, w1 is an approximation by least squares to the pure spectrum of analyte 1, similar to the

78
one performed in CLS assuming the presence of a single analyte in the calibration. In contrast to PCR,
in this step we appreciate the introduction of information concerning the calibration concentrations in
the calculation of the first factor.
Step 2. We continue with the assumption that only anlayte 1 is present, and calculate the contribution
of the first factor w1 in the calibration mixtures. This "concentrations" form the vector t1. Notice that if
indeed a single component occurred in the samples, steps 1 and 2 would be identical to a CLS model.
In the presence of more than one component, PLS deviates from the classical method of analysis.
Step 3. The regression coefficient relating the score t1 calculated in step 2) with the calibration
concentrations is estimated.
Steps 4 and 5. These steps ensure that the subsequent vectors ta and wa will be orthogonal to each
other. For this purpose, vectors pa, called loadings, are calculated. These vectors, in contrast to PCR,
do not explain the spectral variance in the matrix X, but represent an attempt to explain this variance,
and at the same time to correlate the scores ta with the concentrations yn.
In the PLS calibration phase, the vector of regression coefficients vn is obtained, whose elements
(v1,..., vA) are estimated at each of the A steps of the iterative algorithm.
In the prediction step, the regression coefficients are employed to estimate the analyte concentration in
the new sample. The previous step, as in PCR, is to obtain the sample scores, which is done with the
aid of the matrices W and P:
tA = (WTP)–1 WT x (11)
T
yn = (vn) tA (12)
Returning to the above mentioned literature work, notice that it is not possible to determine Pr, Nd or
Sm directly using a univariate calibration graph, and multivariate methods should be employed. The
comparison of the results provided by neural networks and PLS gives the results shown in the
following table:

This latter table shows the mean square errors (RMS) and relative errors (RSEP%) for the three
studied elements, according to several algorithms. The first one is PLS, as was previously described
and the following four are different variants of neural networks. The best results are obtained using a
neural network known as BP-SC, meaning back-propagation (BP) and single-component (SC). For
more details, see the literature work.

79
Bibliography
Books
1. D. L. Massart, B. G. M. Vandeginste, L. M. C. Buydens, S. De Jong, P. J. Lewi and J. Smeyers-
Verbeke, Handbook of Chemometrics and Qualimetrics, Elsevier, Amsterdam, 1997.
2. R. G. Brereton, Chemometrics. Data Analysis for the Laboratory and Chemical Plant, Wiley,
Chichester, 2003.
3. H. Wold, Estimation of principal components and related models by iterative least squares, en
Multivariate Analysis (Ed., P.R. Krishnaiah), Academic Press, NY, 1966.
4. J. Zupan and J. Gasteiger, Neural networks in chemistry and drug design, Segunda edición, Wiley-
VCH, Weinheim, 1999.
Articles
1. E. V. Thomas and D. M. Haaland, Partial least-squares methods for spectral analyses. 1. Relation
to other quantitative calibration methods and the extraction of qualitative information, Anal.
Chem. 1988, 60, 1193-1202.
2. F. Despagne and D. L. Massart, Neural networks in multivariate calibration, Analyst 1998, 123,
157R-178R.

Alejandro C. Olivieri
Departamento de Química Analítica
Facultad de Ciencias Bioquímicas y Farmacéuticas
Universidad Nacional de Rosario
Suipacha 531 S2002LRK. Rosario, Argentina
aolivier@fbioyf.edu.ar

80
 4

Total Reflection X-ray Fluorescence

Analysis - Instrumentation

Peter Wobrauschek

Abstract
Based on the fundamental physics of x-ray total reflection the development and advantages of working
in this special excitation geometry for energy dispersive x-ray fluorescence analysis are shown. The
many fields of application are listed and some special applications reported in detail. Finally the new
developments in the instrumentation mainly on the source and detector side including x-ray optics are
discussed. Some commercially available instruments for total reflection x-ray fluorescence analysis
are presented. A sketch about the mathematical procedures to determine concentrations from the
measured spectral intensities are given at the end.

Introduction
X-ray fluorescence analysis (XRF) is a powerful analytical tool. This includes the capacity to detect
almost all elements of the periodic system from Beryllium to Uranium. Even the highest Z elements of
the Actinides can be detected. Quantitatively the dynamic range covers several orders of magnitude so
ultra trace element levels to major elemental concentration can be determined. In terms of detection

81
limits the levels of femtogram (E-15 g) absolute detectable masses under optimized excitation-
detection conditions can be reached. So these features maybe topped with other properties as rapid
analysis time and simultaneous detection of the elements present. In some applications non
destructiveness is of importance while dealing with precious substances of cultural values from fine
arts also in cases of forensic investigations if only small amounts are available. Here one considers
energy dispersive XRF (EDXRF) which is perfectly suited for these investigations.

The above statements are emphasizing the analytical power and in addition to these arguments one
could add, to point to the revival of X-ray fluorescence analysis, the large number of applications. The
applications range from the interesting field of medicine, techniques and environment to forensic, fine
art, extra-terrestric investigation and fundamental research. All in all starting from the conventional
excitation geometries where excitation and detection is at angles of 45° to the sample surface a lot can
be achieved. With new ideas and modifying the physical properties of the primary radiation e.g.
monoenergetic – linear polarized – high intensive – radiation and together with effects based on
fundamental x-ray physics like total reflection will lead to arrangements in excitation - detection
geometries and an analytical superb output regarding low detection limits. In this contribution mainly
the method of TOTAL REFLECTION X RAY FLUORESCENCE ANALYSIS (TXRF) suggested by
the Japanese group Y.Yoneda and T.Horiuchi in 1971 and then continuously developed since 1974 by
P.Wobrauschek, H.Aiginger, H.Schwenke , J Knoth and R Klockenkaemper will be presented. The
relevant literature is cited (1,2,3,4,5,6,) and in the special issues of Spectrochimica Acta dedicated to
the TXRF Conferences (7-15) with exception of 1995. In addition many groups from industry and
university are using TXRF for interesting application and research making use of the advantages
described in the forthcoming text.

Figure 1. Comparison among conventional and Total reflection mode of excitation (design and
preparation by courtesy of S.Pahlke)

In Figure 1 the experimental setup of conventional EDXRF and TXRF is schematically shown. TXRF
is basically an energy dispersive analytical technique, the main difference to conventional EDXRF is
not the source or the detector but the geometry of excitation. The incident radiation in TXRF is a fine
collimated almost parallel beam with dimensions of 8mm wide and a height of only 50 µm. This
narrow beam impinges at an angle below the critical angle of total reflection (1.8 mrad or 0.1 degrees)
on the surface of a flat polished material which serves as sample carrier. It may happen that as in the

82
case of a Si wafer, the wafer itself is the sample directly and the analysis of the wafer surface is the
analytical task, which can be performed then immediately. In particular the requirements of the wafer
industry in respect of sensitivity and low detection limits described in the metrology roadmap for
process and tool development and manufacturing of electronic devices was pushing strongly for TXRF
as analytical tool in this field (16). In the case of chemical analysis of any sample as from
environment, medicine, technical products, the procedure is different. The sample has to be transferred
in liquid form by chemical digestion procedures. A small volume of the dissolved sample is taken by a
pipette with a volume of 1 – 20 µl. The acidic or aqueous solution is dropped in the center of the
reflector and dried by infra - red heating, hot plate or in vacuum. Recently the collection of fine dust
and also aerosols (17,18,19) directly on the sample carrier by multistage samplers and directly
analyzing the minute masses collected by TXRF was successfully proven in large scale experimental
series in different parts of the world. As one recognizes, the reflector serves for 2 tasks to be the
reflecting medium for x-rays and sample carrier as well. To understand the operation principle of
TXRF a short look into the theory follows.

The theory of x-ray total reflection is based on the phenomenon that at an incident angle smaller than
the critical angle the primary beam is reflected (20). The beam reflects from the surface at the same
angle than the incident one and is having almost the same intensity than the primary beam (total
intensity is reflected), except of a small portion which is refracted and penetrates into the reflector
medium. This evanescent wave loses intensity exponentially the deeper it penetrates. In Figure 2 the
fundamental formalism of x-ray total reflection is shown, based on the Fresnel formalism and the
complex index of refraction for x-rays n which is given below.

Figure 2. Sketch of the theoretical condition s for x-ray total reflection (courtesy C.Streli)

The terms of the complex index of refraction can be divided in the real and imaginary part, where
delta is described by the atomic structure and beta is describing the atomic photoelectric absorption
cross section. So the relation of the index of refraction and the scattering factors of the individual
atoms is given by f1 and f2 tabulated (21). The effective result for the XRF experiment in total
reflection geometry is a drastically reduction of the scatter background in the measured spectra. This is
due to the high reflectivity larger 95% and the narrow zone inside the reflector where x-rays are
penetrating and perform interaction processes as scatter and excitation of the substrate material (Si-K
in the case of a quartz reflector). The physically transferred energy into the reflector leads to resulting
scatter peaks and fluorescence signals from the reflector material in the measured spectra and can be

83
calculated using this formalism. This signal stems significantly only from a few nm below the surface
of the reflector and is called penetration depth and is sketched in

Figure 3. Detailed look in the near surface region during total reflection of x-rays

From this detailed look it is easy seen that the second important advantage besides background
reduction is the fact that the excitation of the sample is done twice, by the primary beam and the
reflected beam as they both pass through the sample. A summary of advantages while measuring in
TXRF is listed in Table 1.

• Double excitation by direct and reflected beam

• Almost no penetration of the primary radiation into the substrate resulting in low background

• Large solid angle as the detector can be placed close to the reflector surface

• Conclusions: S/B improved, DL improved

• Femtogram levels, pg/g Concentrations, E8 atoms/cm² on wafer surfaces detectable

Table1. Advantages of TXRF

Environment:
water: rain, river, sea, drinking water,

waste water.

air: aerosols, airborne particles, dust,

fly ash.

soil: sediments, sewage sludge.

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 plant material: algae, hay, leaves, lichen, moss, needles roots, wood.

foodstuff: fish, flour, fruits, crab, mussel, mushrooms nuts, vegetables,

wine, tea.

various: coal, peat.

Medicine / Biology / Pharmacology:

body fluids: blood, serum, urine, amniotic fluid.

tissue: hair, kidney, liver, lung, nails, stomach, colon.

various: enzymes, polysaccharides, glucose, proteins, cosmetics, bio-films.

Industrial/Technical applications:
surface analysis: Si-wafer surfaces,

GaAs-wafer surfaces

implanted ions depth and profile variations

thin films single layers, multilayers

oil: crude oil, fuel oil, grease.

chemicals: acids, bases, salts, solvents.

fusion/fission research: transmutational elements in Al + Cu, Iodine in water

Mineralogy:
ores, rocks, minerals, rare earth elements.

Fine Arts / Archeological / Forensic:

pigments, paintings, varnish.

bronzes, pottery, jewelry.

textile fibers, glass, cognac, dollar bills, gunshot residue, drugs, tapes, sperm,

finger prints.

Table 2. List of known applications for TXRF from literature

From the list of applications in Table 2 one can imagine the wide field of interdisciplinary activities
which can be developed and performed using TXRF (22, 23, 17-19, 24, 25, 26, 27, 28, 29, 16, 30, 31,
32, 41, 42, 43). For completeness the modern applications in nanotechnology where layers and
implants are characterized should be mentioned where the angle dependence of the fluorescence signal

85
is the major signal leading to the information about thickness distribution and elements present in the
sample layer (33, 34, 35, 36, 37, 38,39, 40)

Instrumentation in TXRF
As in all x-ray spectrometers the three components of importance are

Source – Sample – Detector

and here the description of the instrumentation is following this arrangement of components thus the
source is discussed first.

Sources of x-rays for TXRF are mainly x-ray tubes, ranging from low power 50 W (www.oxford.xtg)
to high power standing anodes 3000W and finally rotating anodes up to 18 kW. All is done to get a
high flux of photons onto the sample. There is the ultimate source with best properties of having a
natural collimated beam characteristic, an extreme flux and a linear polarized beam, this is
SYNCHROTRON RADIATION (SR). Even it is difficult to get access to SR the results achievable
show the importance of this effective combination between source and TXRF where ultimate low
detection limits of femtogram levels have been reached.

X-Ray Sources

Standing anode diffraction tubes:

Anodes: Mo, W, (Rh, Cr, Cu, Ag)
Focal size: 0.04 x 8 mm2 60 kV, 2 kW
0.04 x 12 mm2 60 kV, 3 kW
Au: Focal size: 0.1 x 10 mm2 100 kV, 0.9 kW
Windowless tubes:
Cu- anode, Cu-L= 0.9 keV 20 kV, 750 W
Si anode, Si-K= 1.74 keV, 20 kV, 500 W
Wanode: W-M= 1.74 keV
Rotating anode:
Anodes: Cu, Mo, W
Focal size : 0.05 x 10 60 kV, 18 kW
Synchrotron radiation:
High brilliance
Natural collimation
Linear polarization
Wide spectral range – tunability
Fs laser driven X-ray source:
high harmonics generation , E up to 1 keV

Table 3. List of x-ray sources for TXRF including special types

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A listing of classical and also some exotic sources for very special applications are listed in Table 3.
The beam from an x-ray tube is unpolarized and has a spectral distribution of continuous and
characteristic radiation. In many cases a monoenergetic excitation is the preferred one because the
background is optimally low as only scattered photons from the one energy are present and will appear
as two lines elastic and Compton scattered in the spectrum. Efficient monochromators are nowadays
available by new structures named multilayers with reflectivities in the range up to 80% of the
characteristic radiation of the anode material of choice. Typically in use are combinations of layered
structures made of W-C, Ni-C, Mo-Si with a d spacing of 2-3 nm. Thus they are adjusted to fulfill
Braggs equation and the spectrum is modified to monoenergetic. These ML are commercially
available from many producers worldwide. Using modern technology to bend these structures it is
possible to design x-ray optical components focusing with small divergence the beam and increasing
the flux of photons in the focal spot which is designed to be on the sample position. The result is an
increase of primary intensity on the sample several times more than with the unfocussing flat
multilayer. These optical components are commercially available and can be inserted in the beam path
of the TXRF spectrometer to improve further detection limits (44).

Figure 4. Flat multilayer in beam path of a TXRF spectrometer for Si wafer surface analysis

Typical examples can be seen for these developments in the sketches of the beam path in the series of
Figures 4, 5, 6 A considerable improvement of the flux achieved on the sample spot leads to an
improvement of detection limits. A factor of 1.7 could be reached and is stated in Figure 6.

87
Figure 5. Flat multilayer in beam path of a TXRF spectrometer for Si wafer surface analysis

Figure 6. 2 multilayers in the beam path resulting in lower background conditions

88
A new approach is also done using a double curved Si crystal to focus in both planes to a high intense
spot used for exciting the sample in TXRF geometry (45) reaching with a low power tube 40 W
detection limits for Br of about 40 fg.

The x-ray beam impinging on the sample is modified in the way described above and the reflector
which is the substrate is the next component in the instrumentation to be presented.

The reflector materials used for this technique are manifold. They must fulfill only to have a polished
flat surface which is described by mean roughness in the range of 1 nm and a flatness of l/20. In this
requirement l = the wavelength used in the optical test procedure and is in the range of 572 nm. One of
the advantages is the multi use of the reflectors after a careful cleaning procedure and testing the
blank. This inherently avoids also memory effects of the analytical instrument as nothing is left from
the previous sample.

•Reflector materials

•Quartz Silicon Germanium

•Sapphire Niobium Tantalum

•Plexiglass (Perspex) cheap single use

requirements:•Surface quality: flatness l/10 l=572nm

•Roughness: ~ nm region

Table 4. Listing of reflector materials and requirements for surface quality

Samples and Sample Preparation

The samples can be from many scientific disciplines and thus the physical chemical state is different.
The best suited sample for TXRF is in liquid state –either in aqueous or acidic solutions – so if solids
or powders are to be analyzed these samples must be transferred to the liquid state. Some steps of the
sample preparation were already mentioned earlier in this paper and a scheme of suggested methods is
shown in Figure 7.

The presented procedures are typically but can be of course adapted to the sample type and the world
of chemistry is fully open to new ideas given in detail in the respective literature.

Samples

The sample preparation was discussed by several authors and many publications deal with special
methods.(46, 47, 48, 49, 50, ) Even so the complex subject leaves a lot of ideas open to get the sample
prepared in a way that the elements present can be detected even if they are at lowest concentrations.
Preconcentration methods or selective enrichment techniques by chelation or electrochemically (51)
can be introduced to reach masses present on the sample reflector in the range of absolute pg from
very low concentrated samples e.g. sea water where also the salt extraction is an important preparation
step.

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 Figure 7. Schematics of sample preparation for TXRF ( Courtesy A.Prange)

Detectors

In the past years a breakthrough in the development of energy dispersive detectors has been made. The
typically semiconductor detectors made of silicon with Lithium drift or hyperpure Germanium, Si(Li)
or HP Ge in the well established p-i-n type structure have got a competing technology in the way the
charge collection is done, the sideway or drift technology. The Si(Li) pin detector operates as can be
seen in Figure 8

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 Figure 8. PIN type energy dispersive detector

The charges are created by the interaction of the incoming x-rays with the Si crystal. The number of
electron – hole pairs are proportional to the energy of the absorbed photon and give rise to a charge
pulse with a certain pulse height. The charges are attracted by the respective electrode and collected in
a charge sensitive preamplifier stage and move across the detector volume. The new developed
technique of sideways collection is shown in Figure 9.

91
 Figure 9. Principle of the Si drift detector (52)

The main features are the fast signal processing leading to high throughput of several 100.000 cps
range. The low leakage current levels lead to excellent energy resolution from 125 to 150 ev @5.9 kev
and results from the reduced capacity of the structure of this detector compared to the pin type (52).
The operation at temperatures close to -10° C allows electrically cooling by Peltier effect. This means
no liquid nitrogen cooling with the bulky dewar and consumption of 1l/day required. The sensitive
volumes of these detectors are from 5, 10 to 50 mm², test versions up to 100 mm² are reported, the
thickness ranges from 300µm to 350µm thus the efficiency is a point of consideration if K-lines from
higher Z elements should be measured. The detectors are small in physical size and can be mounted in
any position required from the experimental side.

Figures 10, 11 show the photographs of this modern detectors of Si(Li) compared to Si drift detector
and the size can be estimated from the snout length of 100 mm in on case.

92
Figure 10. Conventional SI(Li) detector with LN2 cooling and dewar (www.gsinst.com)

Figure 11. Compact designed Si drift detector , finger length of 100 mm

93
Instruments for TXRF on the Market

The instrumentation for experiments in TXRF can be performed either with a combination of the
relevant adjustment components as angle by a rotator and position by a translator stage and a reflector
holder on top of the rotator or taking a product from the market. Depending if the basic
instrumentation of x-ray source and detector plus electronic is available simple attachment modules
can be purchased. A low cost unit developed in the mid 1980 is the WOBI module from the author
which is distributed worldwide for TXRF if a smaller number of samples per day has to be analyzed.
Stable and robust mechanical components, no electronics for adjustment and sample change required.

Figure 12. Two versions of the TXRF attachment module, hanging or standing tube

As spectral modification unit a high energy cut off reflector in the beam path is foreseen, which can be
replaced by a multilayer monochromator easily to get an upgrading.

Another interesting solution using the combination of an aircooled low power tube with a Peltier
cooled Si drift detector is the portable spectrometer from Intax Berlin PICOTAX.

94
 Figure 13. Portable TXRF spectrometer (53)

The modern layout of PICOTAX includes also a sample changer in the last version .

A TXRF equipment with high power 2 kW Mo anode tube and sample changer but LN2 cooled
detector with 20 mm² area is from ITALSTRUCTURES and shown in figure 14 and more details are
available in the web (54).

Figure 14. TXRF spectrometer with sample changer for 12 samples high power tube, LN2 detector
(www.Italstructures.com)

Finally a TXRF spectrometer allowing the measurement also of the light elements from Na upwards is
presented from the Atominstitut Vienna x-ray team (55). It is a combination of sample changer inside

95
a vacuum chamber with a high power Mo anode 2kW x-ray tube with monochromator unit directly
attached to the exit window of the tube housing. The Peltier cooled detector in use has 10 mm² and
145 eV/5.9 keV resolution and is mounted downlooking in the vacuum chamber through an O-ring
sealed vacuum feedthrough.

Figure 15. TXRF spectrometer with vacuum chamber high power tube and Peltier cooled detector
(details: www.ati.ac.at)

To demonstrate the analytical performance of this spectrometer named WOBISTRAX a spectrum

including information about achievable detection limits of this system is shown in Figure 16.

96
 Figure 16. Typical spectrum measured with WOBISTRAX from a single element Rb sample

Low background and high sensitivity are main features of this TXRF system which reaches detection
limits of 700 fg in this setup. No air scatter and no absorption in air as well as no Ar K line in the
spectrum are straight forward features.

Quantitative TXRF

The experimental output in TXRF should be a spectrum showing nicely separated peaks of the
elements found in the sample and identified by their characteristic line energy. The qualitative analysis
seems to be very rapid nevertheless has some pitfalls. One has to regard the possible line interference
due to the physics and the electronic energy levels of the atom involved. These problems can be
mostly solved searching for the line series of the element.

By computer codes and line fitting due to energy and emission probability this sort of problems can be
solved rapid and elegant. Some detector artifacts as escape peaks and sum peaks create artificial peaks
in the measured spectra but can nowadays routinely recognized and will not lead to failures in the
spectrum interpretation. More complicated is the quantitative evaluation in the general case of a thick
sample where both absorption and enhancement effects must be considered to get a result which is
accurate. The conversion of measured intensities into concentrations is one of the most important steps
in the analytical task using XRF. In the special case of TXRF the complications are rather completely
stripped off as the approach for the thin film sample can be applied which leads to a simple and linear
relation between intensity I and concentration C of the element considered.

97
•White and characteristic excitation:

•Theoretical calculation needs primary spectral distribution

•Absorption correction A = 1 for thin film approximation

Table5. Formalism for intensity- concentration relation

In table 5 the formalism to calculate the intensity – concentration ratio is shown and the parameters
presented are

I…Intensity of the characteristic K- line from element i

Integral over the spectral distribution for the primary spectrum folded with the respective photoelectric
absorption cross section for the K shell taken over the energy range from the absorption edge to the
max energy found, then follow the other parameters as they appear in the formula fluorescence yield,
emission probability, detector efficiency, absorption correction in the sample A, which is 1 for TXRF
as the thin film approximation is valid.

Absorption in the air path f between sample and detector with f = 1 in vacuum, G geometry factor, m
mass of the sample and Ci the concentration of element i. This result can be simplified further by
taking the calculating the intensities for 2 elements which is shown in Table 6 where also
monoenergetic radiation is assumed.

In the case of using any type of spectral distribution e.g. full continuous plus characteristic or
continuous spectrum with high energy cut off and characteristic or monoenergetic by a multilayer it is
convenient to measure a set of standards with known concentration of all elements. This standard is
also including the internal standard element and one can create in this way a sensitivity calibration
based on experimental data only. Modern more sophisticated software packages include the
calculations based on theory for the spectral distribution and applying these data the calculation can be
completely performed.

Intensities of element i and j and forming the ratio cancels Io, G and m

Table 6. Intensity – concentration for two elements and monoenergetic excitation

This is finally combined as ratio and one sees that G and Io are canceling leaving the simple relation
which is shown in Table 7

98
Ratio between 2 elements with possible extension to more elements

Table 7.

which leads to the final result assuming to have added one element of known concentration to the
sample which is called internal standard. The final result is shown in Table 8.

Table 8. Calculation for the concentrations from measured intensities and sensitivity values obtained from
theory or by measuring multielement standards with chosen added internal standard

99
Determination of the sensitivities S can be done by theoretical calculations or by experimentally
determined S values for each element in a multielement standard solution with the internal standard
included. Table 9 shows the formalism in detail and is basis for calculating the concentration of
unknowns prior to a determination of the sensitivities.

Table 9.

Software packages as AXIL (56) and WINQXAS (57) which are shareware and can be downloaded
from the IAEA homepage are widely used to calculate the intensities respectively the number of
counts measured in a certain time interval with the spectrometer. Thus the net counts are available for
all measured elements and after the calibration of the spectrometer the direct calculation using the
software of unknown elements can be done using the simple quantitative analysis and the subgroup for
calculation in TXRF regression of count rates vs. concentration. A typical result file where element,
line series, counts and concentration are given is displayed in Table 10.

100
 Table 10. Result file for TXRF analysis of a 100µg/g multielement standard solution

Conclusions
TXRF turns out to be a well established analytical tool and has found in some applications like Si-
wafer analysis and water analysis a unique position as practical no sample preparation is required. The
sensitivity achieved is superior compared to conventional EDXRF spectrometers as the fluorescence
signal is doubled because the primary and the reflected beam are used for the excitation of the sample.
The fact that the background is drastically reduced leads to an improvement of the detection limits
several orders of magnitude e.g. µg/g in conventional systems versus ng/g in TXRF. With special
sample preparation techniques even pg/g concentration levels maybe detected. In the routine day by
day work the TXRF system have proved to be reliable and stable. The TXRF equipment from all
commercially producers gives results which are precise and accurate and the software packages are
user friendly and have output files storing all relevant data in order to fulfill the ISO qualifications.

Available TXRF spectrometers range in price from low cost attachment modules to complete systems
which are in the range of 70.000 - 100.000 EURO. Another important feature are low running costs as
only electrical power supply and only in some cases LN2 must be foreseen.

101
References
1 Y. Yoneda, T. Horiuchi , Review of Sci.Inst. 42(7)1069(1971)
2 P. Wobrauschek, H. Aiginger , Anal. Chem. 47(6)852(1975)
3 J. Knoth, H. Schwenke , Fresenius Z. Ana. Chem. 291,200(1978)
4 R.Klockenkämper, Total Reflection X-ray Fluorescence Analysis, John Wiley &Sons, Inc.
(1997)
5 R.Rieder, P.Wobrauschek, W.Ladisich, C.Streli, H.Aiginger,S. Garbe, G.Gaul, A.Knöchel,
F.Lechtenberg, Nucl. Instr. Meth.A355,648(1995)
6 C.Streli, P.Wobrauschek, W.Ladisich,R. Rieder, H.Aiginger, R.Ryon, P.Pianetta, Nucl.Instr.
Meth. A345,399 (1994)
7 Spectrochimica Acta B44 (?), 1989
8 Spectrochimica Acta B46 (10), 1991
9 Spectrochimica Acta B48 (2), 1993
10 Adv.X-ray Chem.Anal.26s,(1995)
11 Spectrochimica Acta B52 (7), 1997
12 Spectrochimica Acta B54 (10), 1999
13 Spectrochimica Acta B56 (11), 2001
14 Spectrochimica Acta B58(12), 2003
15 Spectrochimica Acta B59 (8), 2004
16 L.Fabry, S.Pahlke, L.Kotz, P.Wobrauschek, C.Streli, Fresenius J Anal.Chem. 363:98-102
(1999)
17 B.Schneider, Spectrochim. Acta 44B, 519 (1989).
18 Vilhunen, VonBohlen, Schmeling, Rantanen, Mikkonen, Klockenkämper, Klockow,
Mikrochim.Acta 131,219 (1999)
19 J. Injuk, R. Van Grieken, Spectrochim. Acta Part B 50, 1787 (1995)
20 P.Kregsamer, Spectrochim. Acta 46B, 1333 (1991)
21 At.Dat Nucl,Data Tables 54, 181 (1993)
22 A.Prange and K.Kremling, Marine Chemistry 16 ,259 (1985)
23 R.P.Stössel and A.Prange, Anal. Chem. 57,2880 (1985)
24 Török, Labar, Schmeling, VanGrieken, Anal. Chem. 70(12), 495R(1996)
25 M.Carvalho, M.Barreiros, M.Costa, M.Ramos, M.Marques, X-Ray Spectrom. 25, 29(1996)
26 Liendo, Gonzlez, Castelli, Gomez, Jimenez, Marco, Sajo-Bohus, Greaves, Fletcher, Bauman; X-
Ray Spectrom., 28, 3, (1999)
27 T. Capote,L.M. Marco,J. Alvarado,E.D. GreavesSpectrochim. Acta 54B,(10), 11463-1468,
(1999)
28 L.M. Marco,J. Alvarado,E.D. GreavesSpectrochim. Acta 54B,(10), 1469-1480, (1999)
29 Greaves, Marco Parra, Rojas, Sajo-Bohus, X-Ray Spectrom. 29,349-353 (2000)
30 V.Penka and W.Hub, Spectrochim. Acta 44 B, 483 (1989).
31 W.Berneike, J.Koth, H.Schwenke and U.Weisbrod, Fresenius Z. Anal. Chem. 333, 524 (1989).
32 M.Schuster,Adv.X-Ray Anal. 34,71 (1991)
33 W.W. Van Den Hoogenhof and D.K.G. De Boer, Spectrochim. Acta 48B, 277 (1993).

102
34 H.Schwenke, R.Bormann, J.Knoth and A.Prange, Spectrochim. Acta 48B, 293 (1993).
35 H.Schwenke,J.Knoth, Adv.X-ray Chem.Anal.26s, 137(1995)
36 Tsuji, Sato, Hirokawa, Material Trans. JIM 37(3), 295 (1996)
37 Schwenke, Knoth, Fabry, Pahlke, Scholz, Frey, J.Electrochem. Soc. 144(11), 3979 (1997)
38 D.K.G.de Boer, W.W.van den Hoogenhof, Adv.X-Ray Anal.34,35 (1991)
39 D.K.G. De Boer and W.W. Van Den Hoogenhof, Spectrochim. Acta 46 B, 1323 (1991).
40 D.K.G. deBoer, X-Ray Spectrom. 18, 119 (1989).
41 R.Klockenkämper, A.vonBohlen, L.Moens and W.Devos, Spectrochim. Acta 48B, 239 (1993).
42 T.Ninomiya, S.Nomura,K.Taniguchi, S.Ikeda, Adv.X-ray Chem.Anal.26s,9 (1995)
43 A.Prange, U.Reus,H.Böddekker, R.Fischer,F.P. Adolf, S.Ikeda, Adv.X-ray. Chem.Anal.26s,1
(1995)
44 P.Wobrauschek, C.Streli, G.Pepponi, A.Bergmann, O.Glatter, Nucl.Instr.Meth. A, 482,569-572
(2002)
45 Zewu Chen, oral presentation at Denver X-ray Conference 2001, XOS: www.XOS.com
46 R. Klockenkämper, A. von Bohlen, B. Wiecken, Spectrochim. Acta 44B (5), 511, (1989)
47 Dargie, Markowicz, Tajani, Valkovic, Fresenius Z.Anal.Chem. 357, 589(1997)
48 B.Holynska, B.Ostachowicz, D.Wegrzynek, Spectrochim. Acta B 51,769-773 (1996)
49 A. Prange, H. Schwenke, Adv. X-ray Anal. 32,211 (1989)
50 J.Injuk, R.E.VanGrieken, Handbook of X-ray Spectrometry,p.657, Marcel Dekker(1993)
51 A.Ritschel, P.Wobrauschek, E.Chinea, F.Grass, Ch.Fabjan, Spectrochim.Acta B54, 1449-1454
(1999)
52 Homepage from KETEK: www.ketek.net
53 www.rontec.com
54 www.italstructures.com
55 C.Streli, P.Wobrauschek, G.Pepponi, N.Zoeger, Spectrochimica Acta B 59, 1199-1203 (2004)
56 QXAS, IAEA version Seibersdorf,
http://www.iaea.org/OurWork/ST/NA/NAAL/pci/ins/xrf/pciXRFdown.php
57 WINQXAS, IAEA Seibersdorf,
http://www.iaea.org/OurWork/ST/NA/NAAL/pci/ins/xrf/pciXRFdown.php

Peter Woubrauschek
Atominstitut derÖsterreichischen Universitäten
University of Technology
Stadionallee 2. A-1020. Vienna, Austria
wobi@ati.ac.at

103
 5
Total Reflection X-Ray Fluorescence
Analysis of Low Z elements:
Developments and Applications

Christina Streli

Abstract
Total Reflection X-ray Fluorescence Analysis (TXRF) is a special technique of energy dispersive X-
ray fluorescence analysis, offering the advantage of a multielement method with detection limits in
the pg range or at the ng/g level as well as requiring very small amount of sample. The detection
limits in the pg range are only achievable for medium Z elements using standard TXRF spectrometer.
The detection of low Z elements is a critical point of TXRF since the development of the technique. The
x-ray group of the Atominstitut was investigating from the beginning the extension of the element
range of TXRF to the low Z elements. Besides special X-ray sources to excite the low Z elements
efficiently, a detector suitable for the detection of low energy radiation has to be used. On the source
side synchrotron radiation turned out to be best suited for efficient excitation, results from
experiments at SSRL, California and the PTB Beamline at BESSY2 in Berlin are presented. At lab
scale experiments with windowless X-ray tubes and Si as anode material have been performed and
results will be presented. On the detection side, SiLi detectors specially equipped with ultra thin
windows have been used. Also the new developed Silicon drift detectors are suitable to detect light
elements down to Oxygen, if an ultrathin window is used. Applications as the detection of surface
contaminations of Si wafers at ultra trace levels are shown, as well as aerosol filters and lyophilized
biofilms. Furthermore first results of chemical speciation by XANES in TXRF geometry are shown, on
organic compounds on Si wafer surfaces. The determination of thickness and density of C-films on Si
wafers is demonstrated as well as depth profiles from low Z implants in Si wafers.So instrumental
developments as well as possible applications are reviewed.

104
Introduction
There is a lack of analytical methods performing nondestructive and rapid multi-element analyses of
light elements at trace levels[i,ii,]. TXRF (Total Reflection X-ray Fluorescence Analysis) can meet
these requirements [iii,iv, v,vi]. A special spectrometer adapted to the specific problems of the energy
dispersive detection of low energy radiation must be used. This spectrometer is described in detail in
ref. 5. The main component is an energy dispersive SiLi detector. This detector is equipped with a thin
entrance window (300 nm AP1.3™) and meets the requirements for detecting low energy fluorescence
radiation, the FWHM at 5.9 keV was 137 eV. The experiments are performed in a vacuum chamber to
avoid air absorption. The achievable detection limits are mainly influenced by the kind of excitation
source, which should provide a large number of photons with an energy near the K-absorption edge of
these elements (from 200 eV upwards). The determining factor is the integral over the source spectral
distribution multiplied by the photoelectric absorption coefficient (∫ I(E). τ (E).dE ). Since the
absorption coefficient τ (E) drops steadily as the energy E increases above an element’s absorption
edge, X-ray tubes with standard anode materials (e.g. Sc, Cr, Cu) are poor exciters for light elements
as their characteristic emissions are far above the absorption edges. To improve the sensitivity for light
elements it is necessary to use either an X-ray tube which emits intensive characteristic radiation as
close as possible to the absorption edges of the interesting elements or synchrotron radiation with its
continuous spectral range down to low energies (e.g. τAl (5.4keV)= 163 cm2/g,
τAl(1.74 keV)=3440 cm2/g).

Experimental
Different excitation sources have been used with the low-Z vacuum spectrometer.
A standard Cr-anode fine focus tube (1300 W) with a multilayer monochromator was used in
combination with an 8 µm Kapton foil as entrance window in the vacuum chamber.(ref.5).

Figure1. Schema of the low Z TXRF vacuum chamber

The detector used was an OXFORD Premium with Ultra thin window. Figure 2 shows a spectrum of a
NaF sample.

105
 1000

cts/chn
ref31

Si Cr/40/40/100
90 ng Na, 72 ng F, 3 ng Sc

800

Sc

600

Ca

400

Na Cl

200
S
F
O
0
0 1 2 3 4 5
Energy(keV)

Figure 2. Spectrum of a sample containing 90 ng Na and 72 ng F and 3 ng Sc, excited with Cr tube( ref.9)

The same vacuum chamber was used to test a silicon drift detector with ultra thin window. vii The
results were compared with that obtained with the SiLi. Table 1 shows the obtained detection limits.
Figure 3 compares a spectrum from the silicon drift detector with that from the SiLi.
LLD(ng) LLD(ng) S(cps/ng) S(cps/ng)
SiLi SDD SiLi SDD
C 14 0,0048
N 9 0,0084
O 4 36 0,024 0,007
F 2 5 0,07 0,053
Na 0,57 0,77 0,27 0,29
Sc 0,002 0,004 40 33

Table 2. Detection limits (LLD) and Sensitivity (S) exptrapolated to 1000s measuring time, Comparison of
a 30 mm2 SiLi detector with a 10 mm2 Silicon drift detector, both with UTW

cts/chn
2000
Si Ketek Cr scatter
1800 Oxford
1600 Cr/30/40/100
300ng F 322 ng Na 3 ngSc
1400
1200 Na

1000 Sc

800
600
400 F
200
0
0 1 2 3 4 5 6
Energy(keV)

Figure 3. comparison of a spectrum obtained with the SiLi ( Oxford) with that measured with the Silicon
drift detector ( KETEK)

106
Down to F the silicon drift detector provides good results, but N and C cannot be detected.
To improve excitation conditions a windowless Si-anode X-ray tube was constructed and testedviii.The
anode was prepared by sputtering Si on a polished Al plate, reaching a thickness of 20 µm. The tube is
integrated in the vacuum circuit of the chamber and operated at 2x10-5 mbar. Figure 4 shows a
spectrum and Tab. 3 the obtained detection limits.

2000
Si 15 kV 30 mA 100 s
36 ng Na, 30 ng F
Na
LLD(Na)=36 pg
S(Na)=3.8 cps/ng
LLD(F)= 336 pg
S(F)=0.5 cps/ng
counts/chn

1000

O F

0
0,0 0,5 1,0 1,5 2,0
Energy / keV

Figure 4. Spectrum of a sample containing 36 ng Na, excited with the windowless Si anode tube, 15 kV, 30
mA, 100s measuring time (from Ref.8)

Element S(cps/ng) LLD(pg)

F 0.5 336
Na 3.8 36
Mg 5.3 41
Al 4.3 41

Table 3. Detection limits obtained with the windowless Si anode X-ray tube ( from Ref.8)

A new spectrometer was constructed by the X-ray group of the Atominstitut,, called WOBISTRAX.
This spectrometer is equipped with a 12 position sample changer and a 10 mm2 Silicon drift detector
with an 8 µm Be window. The tube is flexible, for the excitation of the low Z element, a Cr tube was
used.ix Figure 5 shows a spectrum of a sample containing NaF, Table 4 shows the obtained detection
limits.

107
 5000

cts/chn
Cr/30/50/100
Jamcham Sc
90 ng Na, 3 ng Sc

4000

3000

Cr-scatter
2000

LLD( Na) = 900 pg

1000 Si

Na Cl
S Ca

0
0 1 2 3 4 5 6
AL1_01.txt

Energy(kev)

Figure 5. Spectrum of a sample containing 90 ng Na, Spectrometer: WOBISTRAX, Cr tube excitation,

KETEK Silicon drift detector, 10 mm2, 8 µm Be window

Element S(cps/ng) LLD(pg)

Na 0.3 900
Mg 1 156
Al 2 42

Table 4. Detection limits and Sensivitiy obtained with WOBISTRAX, Cr tube excitation, KETEK Silicon
drift detector, 10 mm2, 8 µm Be window

The most efficient excitation was achieved using synchrotron radiation due to its higher intensity and
wide spectral range. The experiments have been performed at SSRL, Stanford, California at Beam line
III-3 as well as at the PTB (Physikalisch Technische Bundesanstalt) Beamline at BESSY2.The
detectors used were all SiLi detectors, equipped with ultrathin windows (AP1.3™), but from various
companies. Table 5 shows a comparison of the detection limits achieved with different excitation
sources.
Cr-1300 Wx WL-Si 500 8 SSRLxi PTB-BESSY2xii
C 4000 0.5
N 0.8
O 1800
F 870 336
Na 230 36 0.062 1.3
Mg 180 41 0.042 0.5
Al 120 41 0.3

Table 5. Comparison of achievable detection limits in pg, extrapolated to 1000 s measuing time. Cr-tube
excitation ( 1300 W), Windowless Si anode tube ( 500 W), Synchrotron radiation at SSRL, BL 3-3 and
Synchrotron radiation at PTB-BESSY2

108
Results and Applications
Environmental samples were investigated using the Cr-X-ray tube excitation. Figure 6 shows the
spectrum of a sample of 10 µl water (NIST 1643c, standard reference material), the concentration of
Na and Mg are in the range of 10 ppm.

1000

cts/chn
Si Ca GR3.txt

NIST 1643 SRM 10 ul

Cr/30/40/100
800

600

Sc= int.std. Cr-

400 scatter
K

200

O Mg P
S
Na Al Cl

0
0 1 2 3 4 5 6
Energy(keV)

Figure 6. 10 µl of NIST water ( 1643c), excited with Cr-tube, 30 kV, 40 mA, 100 s. Sc was used as internal
standard

Figure 7 shows the spectrum of a lyophilized biofilm, Figure 8 the spectrum of the leachate of an air
filter. The sample was leached and the aerosols removed with ultrasonic. Cr tube excitation, Sc was
used as internal standard. (from Ref.10)

1000
cts/chn

Si Ca GR3.txt

Liophylized biofilm
Cr/30/40/100
800

600

K
Sc= int.std. Cr-
400 scatter

200
Al S
Mg P
Cl
O

0
0 1 2 3 4 5 6

Energy(keV)

Figure 7. Lyophilized biofilm sample, Cr-tube excitation

109
 2000

cts/chn
Aerosol sample Si S Ca Cr NIST.txt

Cr/30/40/100 scatter

1500

1000 K

O Sc= int.std.

500

Al P
Na
Mg Cl

0
0 1 2 3 4 5 6
Energy(keV)

Figure 8. Leachate of air filter, water soluble, Cr tube excitation

TXRF can also be used to determine the composition, thickness and density of thin films on reflecting
surfaces measuring the angle dependence of the fluorescence signal and comparing the results with
theoretical models.
At the PTB Laboratory at BESSY2 thin Carbon films on Si wafers have been investigated. A nominal
5 nm Carbon layer was measured , the thickness was determined to be 5 nm with a density of 2.33
g/cm3 which corresponds to the value specified by the producer. Figure 9 shows the obtained results.,
details can be found in ref.xiii

5 nm Carbon on Si wafer

14000
Carbon cts

C(fitted)
C(measured)
12000

10000

8000

6000

4000

2000

0
0 1 2 3 4 5 6
angle ( deg)

Figure 9. Angle dependence of C signal of a 5 nm C-layer on a Si wafer, measured and fitted results.
Measurements performed at the PTB Beamline at BESSY2 (ref.13)

TXRF can be also combined with XANES in fluorescence mode. The advantage is the possibility of
measuring trace elements. First experiments were performed at the PTB beamline at BESSY2 on
organic compounds on Si wafers. Figure 10 shows the energy scan of NaSCN and NaCNO, which can
be distinguished. Details can be found in Ref.xiv

110
 11
2.0x10
NaSCN
NaCNO
11
1.8x10
11
HC=-N sigma*
1.6x10
HC=-N pi*
11
1.4x10

µx [ arbitrary units ]
11
1.2x10
11
1.0x10
10
8.0x10
10
6.0x10
10
4.0x10
10
2.0x10

0.0
396 398 400 402 404 406 408 410 412 414
photon energy [ eV ]

Figure 10. Fluorescence intensity versus energy for NaSCN and NaCNO(from ref. 14)

Conclusions
TXRF is a suitable method to determine also light elements on trace levels in environmental samples.
The detection limits depend on the excitation source, best are achieved with synchrotron radiation, but
also satisfying results are achieved using a Cr-X.ray tube. Applications range from environmental
samples to industrial thin films and speciation of organic contaminations on Si wafer surfaces with
XANES.

References

i
L.Fabry, S.Pahlke and L.Katz, presented at 6th Conference on TXRF and Related Methods, 10-
14 June, Eindhoven/Dortmund (1996).
ii
L.Fabry, L.Köster, S.Pahlke, L.Kotz and J.Hage, IEEE Transactions on Semicond.Manuf. 9(3),1
(1996
iii
C.Streli, H.Aiginger and P.Wobrauschek, Spectrochim. Acta 48B , 163 (1993).
iv
C.Streli, P.Wobrauschek, W.Ladisich, R.Rieder and H.Aiginger, X-ray Spectrometry, 24, 137
(1995).
v
C.Streli, P.Wobrauschek, V.Bauer, P.Kregsamer, R.Görgl, P.Pianetta, R.Ryon, S.Pahlke and
L.Fabry, Spectrochim.Acta B52, 861 (1997)
vi
C.Streli, P.Kregsamer, P.Wobrauschek, H.Gatterbauer, P.Pianetta, S.Pahlke, L.Fabry,
L.Palmetshofer, M.Schmeling, Spectrochimica Acta 54B, 1433-1441 (1999)
vii
C.Streli,P.Wobrauschek, I.Schraik, Spectrochimica Acta B59, 1211-1213 (2004),
viii
Th.Meinschad, C.Streli, P.Wobrauschek, Ch.Eisenmenger-Sittner, Spectrochim. Acta B58,
2069 (2003)
ix
C.Streli, P.Wobrauschek, G.Pepponi, N.Zoeger, Spectrochimica Acta B 59, 1199- 1203 (2004)
x
C.Streli, G.Pepponi, P.Wobrauschek, I.Schraik, Gy.Zaray, 46th Annual Hungarian
Spectrochemical Conference, Szeged, proceedings, p22-26 (2003)
xi
C.Streli, G.Pepponi, P.Wobrauschek, B.Beckhoff, G.Ulm, S.Pahlke , L.Fabry, Th.Ehmann,
B.Kanngiesser, W.Malzer, Spectrochim.Acta 58B,2105-2112 (2003)
xii
B.Beckhoff, R.Fliegauf, G.Ulm, G.Pepponi, C.Streli, P.Wobrauschek, L.Fabry, S.Pahlke,
Spectrochim. Acta B56, 2073 (2001)
xiii
C.Streli, G.Pepponi P.Wobrauschek, B.Beckhoff, G.Ulm, L.Fabry, S.Pahlke, Th.Ehmann,
B.Kanngiesser, W.Malzer, W.Jark, Spectrochim. Acta B58, 2113 (2003)

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xiv
G.Pepponi, B.Beckhoff, Th.Ehmann, G.Ulm, S.Streli, L.Fabry, S.Pahlke, P.Wobrauschek,
Spectrochimica Acta 58B, 2245-2253 (2003)

Christina Streli
Atominstitut der Österreichischen Universitäten
University of Technology
Stadionallee 2 A-1020. Vienna, Austria
streli@ati.ac.at

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 6

Direct Analysis of Biological Samples

by Total Reflection X-Ray Fluorescence

Lué-Merú Marcó P.

Abstract
The technique of Total Reflection X-ray Fluorescence (TXRF) is well suited for the direct analysis of
biological samples due to the low matrix interferences and simultaneous multi-element nature.
Nevertheless biological organic samples are frequently analysed after digestion procedures. The
direct determination of analytes requires shorter analysis time, low reactive consumption and
simplifies the whole analysis process. On the other hand, the biological clinical samples have often
minimal amounts and routine studies require the analysis of high number of samples. To overcome the
difficulties associated to the analysis of organic samples, specially solids one, different procedures of
sample preparation and calibration to approach the direct analysis are evaluated : 1. The slurry
sampling; 2. Compton peak standardization; 3. in situ microwave digestion; 4. in situ chemical
modification and 5. direct analysis with element as internal standard. The analysed samples were
human brain tissue, serum blood, amniotic fluid, urine and the Bovine liver standard 1577a. The
elements Fe, Cu, Zn, Se, Ca, K and Pt among others were determined. The accuracy was evaluated by
comparison of the results to those obtained after the conventional digestion, element as internal
standard and to the independent technique flame atomic absorption spectrometry (FAAS). No

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significant differences were found between the TXRF and FAAS technique results when the last
technique was used for accuracy evaluation. The precision of TXRF results depended mainly on the
ratio of element level to detection limit and was less than 10% of relative standard deviation (RSD) for
most of the quantified elements. The feasibility of the direct analysis using element as internal
standard, Compton peak as internal standard, slurry preparation and also the alternative in situ
microwave digestion and in situ chemical modification procedures for saline matrix elimination was
demonstrated. The direct analysis of urine, amniotic fluid, serum and brain slurries was performed
using element as internal standard and aqueous sensitivity curves since no matrix effects were present
when Z was higher than 22 (vanadium). The TXRF analysis of brain slurries and serum by Compton
peak standardization is accurate and allows to results with analytical quality comparable to the
technique of FAAS and FIAAS. The use of optimized amounts of the modifier allowed to the cuasi total
elimination of the element chlorine from the amniotic fluid samples, the enhancement of the peak to
background ratio in the low energy region of the spectrum and the simultaneous determination of the
elements K, Ca, Fe, Zn, Rb and Sr. Finally, the method of in situ microwave digestion using the quartz
sample holder in the TXRF analysis of biological samples enhanced the peak to background ratio,
specially for the low energy elements K and S. The method is adequate for the analysis of samples
available only in minimal or scarce amounts, as could be biopsies; Since the solvent is evaporated
during the digestion procedure, the sample is not diluted and the detection capability of the technique
is improved.

1.- Introduction
The importance of the direct analysis lies in the fact that the sample preparation is the slow step of the
chemical analysis process. In the field of atomic spectroscopy, most of the techniques as could be
Inductively coupled plasma (ICP), Atomic Absorption Spectrometry (AAS) and even TXRF [1]
require generally the sample digestion in order to avoid matrix interferences, and in some cases for the
appropriate transport of sample to the atomization and detection system. Additionally, the biological
samples demand strong digestion conditions. The procedures may involve the dilution of the analyte to
undetectable levels.
The TXRF is well suited for the direct analysis at trace levels. The matrix interferences are minimized,
with some advantages as could be its simultaneous and multi-element character, the possibility of
microanalysis and the flexibility for calibration (internal standardization [1], Compton peak
standardization [2,3], extended calibration referred to atomic number and fundamental parameters [4]).
Nevertheless, for the quantitative analysis an adequate presentation of sample is necessary since TXRF
demands the special feature of Thin Layer [5]. This condition is difficult to attain, specially in
biological and organic samples, with associated troubles of scattering and self-absorption. To
overcome the difficulties some procedures are discussed in the literature: [6], microwave-assisted wet
[7] and vapor phase [8,9] digestion, acid extraction [7], ultrasonic acid extraction (8), micro-tome
sections [10,11], slurries [9,12,13], simple dilution [14], Electro-deposition[15,16], Amalgamation
[17,18] , Plasma ashing [19], Hydride generation [20], Microwave-assisted drying [21], and less
frequently alkaline fusion [22]. Some of these procedures are in situ onto sample reflector carried out
for extraction/preconcentration of the analyte, partially minimizing sample manipulation and the
subsequent possibility of contamination [15-19,21,23].
The direct analysis itself or combined with in situ preparation procedures is a suitable alternative for
the quantitative determination of trace elements by TXRF. It implies a reduction of analysis time,
sample handling, minimizes contamination and analyte losses.In this work four methods for the direct
analysis of biological samples were evaluated: 1.- Direct Analysis with element as internal standard 2.-
The method of Compton peak standardization 3.- The in situ microwave digestion of sample 4.- The
in situ chemical modification of the analytes.
Direct analysis
Tissue samples, serum blood, whole blood, urine and amniotic fluid can be analyzed by means of
direct determination of the analytes. For the direct analysis by TXRF must be taken into account that
sample on the reflector accomplish the thin layer condition. The self-absorption and scattering must be

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minimized by an adequate sample preparation (micro-tomes, slurries, dilution, etc. [11-15]) and
drying procedure. High background levels associated to organic matrices must be reduced by using a
monochromator (Multilayer or crystal) [14,25]. The aqueous sensitivity curves can be used for the
quantification after the appropriate verification. It must be tested that the sensitivity ratios are
independent of the sample matrix [13].
For monochromatic excitation, the intensity of a given peak is obtained integrating all infinitesimal
contributions over the excitation area of the source, the sample area and the depth of sample and the
detector crystal [13,14]. The quantification by TXRF demands the addition of an internal standard.
The relationship between intensity and concentration is expressed by Eq. 1

Ni SC
= i i Eq. 1
N s S s Cs

Where, Ni y Ns are the net peaks intensity of the element and the internal standard, Si and Ss are the
sensitivities of the analyte and internal standard and Ci and Cs concentration of the analyte and
internal standard, respectively.
This relation holds also when Ss refers to the Compton peak, if the reflector contribution to scattering
is negligible, as it is the case of thin organic samples. When Compton peak is used as internal
standard, Cs becomes unity. After calibration, the concentration can be deduced form the same
equation, because the intensity ratio is measured in the spectrum and the normalized sensitivity and
standard concentration are known.
Compton Peak standardization
The incoherent or Compton peak of TXRF spectrum is related to the mass of the sample therefore it is
possible to take the area of this peak as an internal standard [2,3].This will make the addition of an
internal standard unnecessary therefore resulting in an easier, faster and less sample-consuming
procedure. Due to the fact that the area of the incoherent or Compton peak is critically dependent on
the sample matrix being analyzed, this method take matrix effects into consideration. Besides for
organic samples the incoherent peak is usually high which means that dispersion caused by the
reflector can be considered negligible.
The dependence of the analytical results on the sample matrix, when the incoherent peak is used as an
internal standard was overcome using a sample doped with know amounts of those elements non
existing, or existing in the sample at negligible concentrations as calibration standards [14,25]. After
the normalized sensitivities are found, a fitting of the curve of sensitivity with a second or third order
polynomial permits us to obtain all the values of sensitivities in the selected range of Z, and to
interpolate the calibration curve to include sensitivity values for the elements of interest in the sample.
The use of the described method of Compton peak standardization is restricted to samples with low
variability in their matrices. This can be tested by determining the ratio of an internal standard to the
Compton peak [14,25] and evaluating the relative standard deviation for a representative number of
samples. The procedure can be applied to serum samples [14,25] and some tissues like brain [14] and
leaves [13]. Amniotic fluid and urine samples i.e. are not suitable since they present a high RSD in the
mentioned ratio.
In situ microwave digestion
The digestion of small quantities of samples may have serious difficulties [26]: lack of
representativity, significant contamination and losses and demand for special equipment and
accessories [27]. Nonetheless, such a procedure could be useful for analysis of samples obtained at
small amounts as biopsy tissues, blood samples from children, etc. To take into account the advantages

115
of TXRF for the micro-analysis and the suitability of the in situ digestion onto the sample quartz
reflector a procedure aimed by microwaves was developed [28]. The procedure was evaluated in terms
of possible improvements in the signal-to-background ratio, because of the direct impact this has on
the detection limits. The recovery rates of the analytes of interest were determined. The micro-
digestion increases the signal-to-background ratio, particularly for low-energy elements, thus
improving the detection limits, while providing higher recovery rates for most of the analytes. An
enhancement of the recovery rate is obtained for elements with low Z, as for example S and K. The
method may well be suitable for analysis of samples of which only scarce quantities would be
available, e.g., biopsy tissues, blood samples from small children, etc., if the contamination and
volatilization problems are controlled.
The chemical modifier concept in TXRF
Although some samples may be analyzed by TXRF as solids [29], slurries [29,30], or liquids [30,31],
without previous treatment, there are certain types of samples which require a proper preparation.
Such is the case, for example, of samples with a high chloride content (e.g. seawater, urine, etc.)
These samples represent a two-fold problem. Firstly, the presence of crystallites after drying of the
sample droplet scatter the radiation coming from the X-ray source, thus increasing the background and
negatively affecting the detection limits. Secondly, the appearance of a high-intensity chloride Kα
peak decreases the likeliness of proper quantification of low-energy lines (e.g. K, Ca, etc.).
To deal with saline samples, complexation with sodium dibenzyldithiocarbamate (NaDBDTC), and
separation by reverse-phase chromatography is a frequently used procedure [32-36]. Electrochemical
pre-concentration directly onto niobium [16] or glassy carbon [17] electrode/reflectors has also been
developed for separation of analytes.
Although the chemical modifier concept is proper of GFAAS, it has been recently applied to TRXF
[18,19,21,22,31]. Most of those works have involved thermal stabilization of volatile elements during
the drying stage of the sample aliquot, but there are also some attempts made to improve the
distribution and thickness of the solid residue left after sample drying on the reflector surface, prior to
analysis by this technique [37,38].
Chloride-containing compounds are regular concomitants in most environmental and
biological/clinical samples. These compounds represented an important source of spectral and
physical-chemical interferences in the early days of the GFAAS technique. Because of its high thermal
stability, sodium chloride (mp 801 oC, bp 1413 oC) [39] cannot be readily eliminated in the pyrolysis
stage of the atomization program. In order to minimize such inconvenience, Ediger resorted to the
addition of ammonium nitrate to promote the formation of ammonium chloride [40]. The lower
boiling point of such compound (subl. 340 oC) [39] would thus permit removal of the interference at
lower pyrolysis temperatures. Though the latter is easily conducted in GFAAS, thanks to the intrinsic
temperature control of the graphite furnace atomizer [41], the same is not applicable to TRXF. For a
given compound to be properly used as a chemical modifier in TRXF, it must meet two main general
requirements: i) alter a physical-chemical property of the system (analyte, matrix and/or the reflector
surface) according to the case; and, ii) leave a thin film [42].

3.- Experimental
3.1.- Instrumental
A TXRF spectrometer designed at the Atominstitut der Österreichschen Universitäten, Vienna,
provided with a molybdenum anode (17.5 keV Ka), operated at 40 kV and 20 mA, as X-ray source,
and Si(Li) detector(180 eV resolution). The carbon-molybdenum multilayer crystal and TlAP
monochromator were used. The spectra were collected in a PC-based multichannel analyzer (Canberra
S-100), with live collection times of 250 s, 500s and 1000 s. Acid-cleaned quartz reflectors (∅= 30
mm) were used for sample deposition.. A ceramic hot plate (CORNING), with a temperature
regulation dial, from "Low" to "High" settings, was used for in-carrier pretreatment of amniotic fluid
samples.

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A METTLER analytical balance (± 0.01 mg), model AE240 was employed for weighing of sub-
samples. A Sharp Carousel Model R-2A48 domestic microwave oven was used for in situ digestion of
the samples. The optimized applied power for sample digestion was experimentally determined and
has a value of 84.4 W when the “Defrost” function of the oven is used. This value is equivalent to 20%
of the full power of the oven.

3.2.- Samples and Standards

Amniotic fluid for direct analysis
The AF samples were obtained from patients during the second trimester pregnancy referred to the
School of Medicine Genetics Department at Universidad Centro Occidental Lisandro Alvarado
(UCLA) in Barquisimeto, State of Lara, Venezuela. In addition, 12 samples were obtained from the
Concepción Palacios Maternity Hospital, Caracas, Venezuela. The samples were extracted by
Amniocentesis. The first mililiters of each sample were discarded. Two disposable tubes used for
centrifugation were filled, each one, with 5 mL of the same AF sample. After centrifuging each
sample at 2,400 r.p.m, the solid residue was separated to carry out cariotype determination and the rest
of the sample was stored at –10 °C for later analysis.
The AF samples were acidified in order to avoid variations of the analyte concentration as a function
of time8. For the TXRF study, 10 µL of a 1000 µg mL-1 vanadium standard solution were added to 250
µL of each AF sample to use the vanadium as an internal standard. From each standardized sample, 10
µL were deposited on a quartz reflector, vacuum dried and irradiated during 1000 s.
For the TXRF calibration, inorganic multi-elemental solutions containing K, Cr, V, Co, Ni, Se and Sr
were prepared from Individual 1000 µg mL-1 stock solutions (Titrisol®, MERCK) to cover a
concentration range from 2 to 100 µg mL-1. For several AF samples, 1 mL of each sample was filled
with a 10 µg mL-1 multi-elemental standard solution containing V, Cr, Co, Ni and Se in a 2 mL flask.
From each one of these prepared standards, a volume of 10 µL was deposited on a quartz reflector,
vacuum dried and irradiated during 250 s. This was done to determine detection limits and evaluate
the use of the Compton peak as an internal standard as well as the calibration with a standard element
added to the sample matrix. For the Zn determination using Atomic Absorption, standards were
prepared from a 1000 µg mL-1 stock solutions (Titrisol®, MERCK) solution covering the concentration
range from 0.1 to 1µg mL-1.
Amniotic fluid using Chemical Modifiers
Five (5) amniotic fluid samples were analyzed after the addition and without addition of the chemical
modifier solution. Individual 1000 µg mL-1 stock solutions (Titrisol®, MERCK) of potassium,
vanadium, manganese, zinc, selenium and strontium, were employed for preparation of multi-
elemental standard solutions. Ammonium nitrate (Riedel de-Häen) was employed as chemical
modifier. Sodium Chloride (Riedel de-Häen) was used for preparation of model saline matrix. A 1000
µg mL-1 nickel solution (Fisher Scientific Inc.) was used for preparation of the internal standard
solution. Nitric acid (Riedel de-Häen) 10% w/V was used for cleansing of quartz reflectors. Distilled,
de-ionized water was employed for rinsing and dilution purposes.
A 5 %m/V ammonium nitrate solution was prepared by direct weighing and dilution with de-ionized
water. A 10 µg mL-1 Ni solution, prepared by dilution with de-ionized water, was used as internal
standard. Aqueous multi-elemental standards (5, 10 and 20 µg mL-1) for calibration purposes were
prepared by serial dilution of elemental stock solutions (K, V, Mn, Zn and Sr) with de-ionized water.
Calibration curves were also made by spiking 900 µL of a pooled amniotic fluid sample with 100 µL
of a multi-elemental standard solution (V, Mn, Zn and Sr) to yield final concentrations of 5, 10 and 20
µg ml-1. Detection limits were determined in the amniotic fluid matrix and aqueous standards plus and
without the chemical modifier as well the calibration curves.
Ten micro-liters of the standard/sample and ten micro-liters of the mixed chemical modifier + internal
standard solution were deposited, in that sequence, on the center of the reflector surface by means of a

117
micropipette. Samples were first dried under an infrared lamp, and then heated on a hot plate until
evolution of white vapors. Once dried, the reflectors were allowed to cool to room temperature in a
laminar flow bench, and then subjected to irradiation in the spectrometer and the corresponding
spectra collected for further analysis (tlive= 250 s). Infrared lamp drying alone was used for analysis of
the samples in the absence of the chemical modifier.
Urine samples by direct analysis
Urine samples from normal individuals (students) were collected in plastic vessels after an overnight
fast. Urine samples taken from patients under treatment with Pt containing drugs in the Pediatric
Service at Instituto Oncológico Luis Razetti in Caracas, Venezuela were collected in plastic vessels,
always 24 hours after administration of the drug in a dose of 100 mg/m2. Samples kept in a freezer at -
10 oC until analysis.
Urine standards were prepared taking 5 ml of urine from normal individuals and spiking it with
appropriate amounts of standard solutions of Pt, Sn, Pb and Co to obtain multi-elemental standards
with concentrations of 1, 10, 30, and 50 µg mL-1 of the elements and a 1:1 ratio of urine and water.
Multi-elemental Aqueous calibration standards were prepared by serial dilution with distilled de-
ionized water from stock standard 1000 µg mL-1 stock (Titrisol®, MERCK) solutions of Pt, Pb, Sn, and
Co. The resulting concentration of multi-elemental calibration standards were 2, 10 and 20 µg mL-1.
Standards in serum and urine matrix were prepared assuming that the platinum content in serum and
urine samples from normal individuals is negligible. The detection limits were determined and the use
of the Compton peak as an internal standard was evaluated as well as the calibration with a standard
element added to the sample matrix in comparison to aqueous calibration.
For measurements, ten micro-liters of each sample or standard were placed onto synsil reflectors and
dried under an IR lamp before irradiation in the spectrometer during a live counting time of 500
seconds.
Serum blood samples
Blood samples from normal individuals (blood donors and students) were collected by standard
venipuncture, after an overnight fast, into disposable syringes. The samples were transferred to
centrifuge tubes, allowed to clot for 30 min. and then centrifuged (2400 r.p.m.). The sera were
separated to individual Eppendorff vials and stored at –20 oC until analysis. Samples from cancer
patients before and under treatment were taken at Servicio Oncológico (BADAN-Lara) following the
same procedure, in order to determine the oligoelement levels. Blood samples for Pt determination
were taken in the Pediatric Service at Instituto Oncológico Luis Razetti in Caracas, Venezuela. These
samples were collected in plastic vessels, always 24 hours after administration of the drug in a dose of
100 mg/m2 .
Individual 1000 µg mL-1 stock solutions (Titrisol®, MERCK) of potassium, vanadium, manganese,
cobalt, zinc, selenium and strontium, were employed to prepare multi-elemental standard solutions for
aqueous K-lines calibration curve. In similar way using Platinum, Tin and lead stock solutions
(Titrisol®, MERCK) were prepared calibration multi-element standards for L-lines calibration. Serum
standards were made taking 1 ml aliquots, and spiking it with appropriate amounts of Pt, Pb, Sn and
Co standard solutions, to obtain standards of 1, 5 and 10µg mL-1 of the elements and a 1:1 ratio of
serum to water [25]. A typical spectrum is shown in Figure 1. For testing accuracy, were prepared
serum and urine samples by the procedure described above, but spiking only Pt and Co. Final
concentrations in these standard were 1, 5, 10 and 40 µg mL-1 of Pt and Co in serum, and 2 µg mL-1of
Pt in urine.
Replicates of two of the samples were prepared to check the precision of the measurements. The
internal standard (Vanadium or Cobalt) was added to a final concentration of 10 µg ml-1 in samples.
Ten µL sample/standard aliquot was dispensed onto a previously siliconized quartz reflector. Samples
were dried using the infrared lamp and irradiated during 500 s of live counting time.
Serum blood samples by Compton peak standardization

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Serum standard samples for K lines calibration were prepared by spiking with known amounts of
stock solutions of V, Cr, Ni As and Sr, 1 ml of sample aliquot. Final concentration of the elements
added was 10 µg mL-1. In similar way, for the L lines calibration known amounts of stock solutions of
Sn, Pt and Pb were added to serum samples, as described in the preceding section.
To evaluate the feasibility of the use of the Compton peak as internal standard [14], the procedure was
repeated for ten randomly selected samples for each matrix. The mean value and standard deviation of
the intensity ratio of the element to the Compton peak was calculated to verify that the organic matrix
does not display evidence of variation from one sample to another.
Calibration was conducted using spiked samples and measuring the analyte sensitivities relative to the
Compton peak. A sensitivity curve is drawn by fitting these values with a second or third order
polynome. The latter permits obtainment of sensitivity values of the elements included in a given
range of Z enclosing the analytes actually present in the samples. The detailed description of the
method, is referred to [14]. In all cases, a 10 µl sample aliquot was dispensed onto a previously
siliconized quartz reflector for irradiation during 500 s. Samples were dried using the infrared lamp.
Measurements by the Flame Atomic Absorption Spectrometry were performed to serum samples, in
order to evaluate the accuracy of the TXRF procedures for the analysis of Zn, Cu and Fe. A
Spectrometer Perkin Elmer model 2100 with background correction using a deuterium lamp was used
for FAAS analysis.
Brain samples
Brain samples from healthy, male individuals, who suffered accidental and instantaneous death were
taken at Morgue of the Hospital Central of Barquisimeto, Edo. Lara, Venezuela. Brains were
dissected, not more than 24 hours after death, and kept at -50 oC until sample preparation. Different
brain sections, such as cerebellum, hypothalamus, frontal cortex, vermix and encephalic trunque, were
weighed and homogenized with de-ionized water with a high speed homogeneizer at 23000 rpm.
Homogenates with a 50-60% w/V (wet weigh) of brain tissue were obtained, and kept at -50 oC until
analysis.
Slurries were prepared following the next procedure [43]. An appropriate aliquot of homogenate is
transferred to a volumetric flask. In this step, the pipette is rinsed with de-ionized water in order to
flush any portion of the sample remaining in the inner wall of the pipette. De-ionized water is added to
the sample, filling approximately half of volume capacity of the flask. When acid addition is
considered, the appropriate volume is added to the flask, depending of the desired acid concentration.
Acid addition, however, must be added in the latest step, otherwise de-naturalization of the
homogenate is observed, with the subsequent formation of particles with unwanted sizes. The volume
is then completed with de-ionized water. The slurries in the flask are treated for 10 minutes in
ultrasonic bath at 25 oC previous to their analysis. Slurries were prepared in concentrations from 2.5 to
24% w/V of brain tissue according to the aforementioned procedure. Nitric acid concentration was
varied from 0 to 5% V/V.
In order to perform the microwave-assisted sample digestion 2 ml of homogenate was transferred to
microwave vessels, and mixed with 5 ml of HNO3 Suprapur®, 65 %V/V (MERCK) and drops of 30
%V/V H2O2 (Riedel de-Häen) Vessels were closed and were subjected to a 15 min step at “Medium
power”, and a final 10 min step at “Maximum power”. A similar procedure was used for digestion of
0.35 g of SRM 1577a Bovine Liver, which was used to check the accuracy of the methodology.
Slurry brain standard samples for K lines calibration were prepared spiking known amounts of stock
solutions of Co, V, Ni and Se to 1 ml of sample aliquot, in a similar way as done with the serum
standard samples. A typical spiked brain slurry calibration sample is shown in Figure 2. It was taken
into account that the samples and standard samples were matched in the sample to water content. Final
concentration of the elements added was 10 µg mL-1. To evaluate the feasibility of the use of the
Compton peak as internal standard, the procedure was done to ten random selected samples for each
matrix. The mean value and standard deviation of the intensity ratio of the element to the Compton
peak was calculated, in order to verify that the organic matrix does not evidence variation, and the
Compton peak, from one sample to another with the same biological matrix.

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To the digested 1577a NBS Bovine liver Standard was added V and Co in the same way as the
samples, to check the accuracy of the Cu and Zn results obtained by the procedure of internal
standardization with an element in the serum and brain.
For all measurements, ten micro-liters of each sample or standard were placed onto synsil reflectors
and dried under an IR lamp before irradiation in the spectrometer during a live counting time of 500
seconds for brain and the Bovine liver standard.
Measurements by the technique of Flow Injection Atomic Absorption Spectrometry (FIAAS) and
Flame Atomic Absorption Spectrometry (FAAS) were performed to brain samples, in order to
evaluate the accuracy of the TXRF procedures for the analysis of Zn and Cu in brain samples.
A Spectrometer Perkin Elmer model 2100 with background correction using a deuterium lamp was
used for FAAS and FIAAS analysis.
Standard reference materials by in situ Microwave digestion
The NIST Bovine Liver standard reference material (SRM 1577a) was used for the development and
evaluation of the methodology proposed. Titrisol®, 1000 µg mL-1 standard solutions (MERCK) were
employed for preparation of aqueous multi-element standard solutions by serial dilution. Suprapur®,
65 %V/V HNO3 (MERCK) and 30 %V/V H2O2 (Riedel de-Häen) were employed for digestion purposes.
A commercial silicon spray brand was employed for siliconization of quartz reflectors.
Approximately 1.1 mg of the corresponding standard reference material was accurately weighed
directly on previously siliconized reflectors. Ten micro-liters of a 10 µg mL-1 Co solution, 20 µl of
concentrated H2O2 and 20 µl of concentrated HNO3 were sequentially deposited on top of the SRM.
The reflector was then placed inside the microwave oven and subjected to microwave radiation for 8
min, using the “Defrost” option. Once finished the digestion step, the reflectors were allowed to cool
to room temperature and dry in a laminar-flow bench prior to analysis by TRXF. For comparison
purposes, 1.1 mg of the SRM 1577a were weighed on the reflector and 10 µL of the internal standard
solution were dispensed on top of the sample. This sub-sample, hereby referred to as “crude” sub-
sample, was dried under an infrared lamp and analyzed as described in the next section.
Calibration curves (relative sensitivity vs. atomic number) for Kα line elements were obtained with
aqueous standard solutions covering the range of atomic number from 17 (Cl) to 34 (Se). A cobalt
solution (10 µg mL-1) was used for internal standardization. Ten micro-liters of the aqueous standards,
followed by ten micro-liters of the internal standard, were deposited on the center of the quartz
reflector and dried under an infrared lamp. The standards and the Bovine liver sample SRM 1577a
were then analyzed by TRXF with irradiation live times of 250 s and 1000 s respectively. The
standards and the Bovine liver sample SRM 1577a were then analyzed by TRXF and the
corresponding spectra were recorded in 250 s and 1000 s. Non significant differences in precision and
accuracy were found using both counting times for the elements reported in this work. A good
statistical is obtained at 250 s of live counting time.

4.- Results and Discussion

4.1.- Direct Analysis
Lower detection limits are allowed when the monochromator (TlAP crystal or C-Mo multi-layer) is
used instead of the cut-off filter if organic samples are analyzed [44]. Although with the TlAP or C-
Mo multi-layer set up, the intensity of the signals are about ten times lower than with the cut off set
up, in the set up using the monochromators, scattering of radiation is minimal, which contributes to the
reduced background in comparison to the cut-off set up. If the Cut off is the only choice, then a filter,
such as Aluminum must be additionally used, as reported by Zarkadas et al. [45].
Eventhough the detection limits using the TlAP or C-Mo multi-layer set-up are lower than those
obtained using the cut-off filter, in both instances the detection limits are generally higher for the
samples than for the aqueous standards, when analyzing serum, urine and amniotic fluid, specially for

120
lower Z elements as potassium and calcium. In the case of brain slurry samples, an uniform thin film
was obtained after drying the sample on the reflector. The detection limits in brain slurry samples were
independent of the matrix, even at high slurry concentrations, and also did not differ from the aqueous
matrix [14].
A possible reason for this difference in detection limits in the serum samples could be the lack of
uniformity of the thin film formed on the synsil reflector after the sample is dried on it. For serum, the
presence of foam causes a formation of an irregular thin film when sample is dried under vacuum. It is
recommended the drying procedure at atmospheric pressure with the IR lamp. In such case lower
detection limits are obtained by a factor of 2.
In the urine and amniotic fluid samples the detection limits are affected by the high saline content,
specially for the low Z elements K and Ca.
It was observed that the matrix does not affect the analyte to internal standard ratio when V and Co
are used as internal standard in brain and serum. The sensitivity curves shown in Figure 3
corresponding to water, urine and serum are similar to those obtained for brain and amniotic fluid, and
also to those reported in previous works [14,46]. This allows to the use of aqueous standard calibration
when urine, brain, serum and amniotic fluid samples are analysed, simplifying the whole process.
Brain samples
Zinc and Cu results in slurry brain samples obtained after direct analysis by TXRF were evaluated by
comparison to FIAAS technique and to digested samples by TXRF. The results of brain analysis by
the different comparison methods are shown in Figure 4. A good agreement was found between Cu
and Zn results for slurry samples and those from corresponding digested samples. Also good
agreement was found respect to the FAAS technique. For the analysis of Bovine liver standard, were
obtained the concentrations of 128 +/- 10 µg ml-1 of Zn and 144 +/- 13 10 µg ml-1 of Cu, being the
certified values 123 and 158 µg ml-1 respectively. This confirms the accuracy of Zn and Cu results in
brain and serum samples by the TXRF method using element (V or Co) as internal standard.
Amniotic fluid
The data to obtain the Detection Limits (DL) values reported in this work were gathered, in general,
using different irradiation times, the DL values were extrapolated to a counting time of 1000 s which
is usually accepted [47]. Figure 5 shows a comparison between the detection limits (DL) obtained
from the analysis of an amniotic fluid sample (solid squares), an amniotic fluid sample diluted 1:1
with de-ionized water (empty triangles and dotted line) and water (solid circles). It is clear that for a
given element, the matrix does not influence significantly the DL at high energies, e.g. for high atomic
number values . However, similarly to urine samples previously analysed [48], the differences in DL
values due to matrix type are more pronounced at low energies as expected from samples with high
salt content [49]. The DL values obtained in this study are consistent with those reported by Greaves et
al. in AF samples analyzed with a TlAP monochromator [44]. In Table 1, a comparison between
FIAAS and TXRF measurements is presented. According to the F test, there is an acceptable
correlation between these measurements at high concentrations with a confidence level of 0.05, F =
61.65 and p = 0.00. However, the results at low concentrations do not show this degree of consistency.
In that case, we obtain F = 6.66, p = 0.026 and a critical F value of 4.84 that is very close to the
calculated value. This inconsistency is the consequence of large statistical uncertainties (≥ 20% )
associated with low counting rates due to low concentrations of the element under analysis.
The peak to background signal ratio is improved with the use of the monochromator Unfortunately,
although the use of a monochromator increases the capacity of the TXRF technique to detect Zn, the
Zn detection limits are still higher than the DL values obtained with FAAS, GFAAS and FIAAS. In
spite of this disadvantage, one of the major benefits of TXRF is the possibility of carrying out a
simultaneous analysis of a large number of elements of clinical interest which, in general, speeds up
the process of analysis. In addition, a major advantage of TXRF is the use of small quantities of the
sample under analysis. This fact enhances the necessity of elimination of the salt matrix for a better
performance of the TXRF technique, as discussed in the chemical modifiers section of this work.

121
Urine samples
The direct analysis of urine samples was carried out using aqueous calibration, since sensitivity was
not affected by the urine matrix, as shown in Figure 3. The main difficulty in the analysis is a
consequence of the high saline content. The detection limits are higher than those of water matrix, as it
is deduced from Figure 5 (empty squares), for elements with Z below 23. However, The elements K
and Ca have relatively high concentration in urine samples, as shown in the Figure 6. The dilution
with water could be recommended, but in this case the analites at 1 µg mL-1 level or less could fall
under detection limit.
Quantification of Pt in urine samples using Co as internal standard ( See Figure 7) and aqueous
standards for calibration produces reliable results if the experimental set up includes the use
monochromator crystal, as it is well discussed in [14,25]. Results are shown in table 2. Precision at µg
mL-1 level is less than 10% of relative standard deviation. The accuracy was also verified, finding a
good agreement between the determined concentration and the spiked amount (as explained in the
section of samples and standards).

4.2.- Compton Peak

Although the brain slurry and serum samples are very complex, their matrices are practically constant
in composition and independent of the individual or the brain section. This is supported by the
estimation of the internal standard to Compton peak ratio in ten replicates, being the relative standard
deviation less than 10% in both samples. This is not the case of the amniotic fluid and urine samples,
having a high variability in their matrices, even for a same individual. This fact was tested by
estimation of the Cobalt to Compton peak ratio in ten samples (Amniotic fluid or urine), being the
relative standard deviation higher than 20%. The sensitivity curves relatives to Compton peak area as
internal standard are shown in Figure 8, for the K lines in serum and brain slurries, and L-lines in
serum matrix. For comparison it was drawn also the aqueous curve using vanadium as internal
standard.
The results of Zn and Cu analysis in brain slurry samples using Compton peak standardization,
compared to element as internal standard, are shown in Figure 9. As deduced from figure and
corroborated by the confidence test, the results by both procedures are in agreement. When Zn results
obtained with V as internal standard are compared to those obtained with Compton peak as internal
standard, using the F correlation Coefficient, at 95% confidence level (p=0.05) F coefficient was 22.7,
being the critical value of F 5.99. In the same way, when using Co as internal standard, the value of F
was 14.95.
Similar results are obtained for Cu analysis in brain samples. There is a good correlation between
results obtained by normalizing to V or Co and results obtained by normalizing to Compton peak, as it
is observed in Figure 9. The confidence test at 95% of confidence level demonstrates that the results
are in agreement. The comparison to the independent technique FAAS is shown in Figure 4. No
significant differences were found between the TXRF and FAAS methods.
As observed for brain slurry samples, in serum samples a good agreement was found between results
obtained by Compton peak standardization and those obtained by standardization with Co or V. The
confidence test of F correlation gave for Fe, Cu, Zn and Se F values of 144, 77, 151 and 112, and an
associated probability of p=0.00 at 95% confidence level (0.05). To Pt results was applied the t-
Student test. The test confirms, that results obtained by both methods of calibration are identical. The
accuracy of TXRF with Compton peak standardization was verified by comparison to the FAAS
technique for the elements Fe, Cu and Zn, using random selected samples (n=40), as described in
reference [47]. In Figure 10 are shown the values obtained for brain slurries and serum by FAAS and
TXRF with Compton peak as internal standard. The accuracy for Selenium was verified obtaining the
percent of recovery in the serum sample spiked with 0.95 µg ml-1 of the element. The percent of
recovery was 93%.

122
The precision was evaluated by three ways: Precision rotating the reflector, precision between aliquots
and precision between replicas, for all the analyzed elements. It was observed that the precision
depended on the concentration of the element and the detection limit. When the element concentration
is closed to detection limit, as is the case of Selenium, the mean precision was about 16% of Relative
Standard deviation, rotating the sample holder, in the aliquots and replicas. For the elements Fe, Cu
and Zn, in both matrices (serum and brain), the precision was around 8% of Relative Standard
Deviation. The levels of these elements were ten times higher than the detection limit. The precision of
Pt results was 10% of Relative Standard deviation for aliquots and replicas.

4.3.- In situ Microwave Digestion

The in situ digestion of small samples (microanalysis) demands careful control of the digestion
conditions in order to prevent contamination, volatilization losses, etc. The reflector surface must be
rendered hydrophobic by an adequate siliconization to minimize the spreading of the sample aliquot.
The contribution of the commercial silicon and reagents to the blank signal was verified for all the
spectra. It was negligible, as reported in reference [29].
The order of addition of the digestion reagents is critical. They must be added in the sequence H2O2
followed by HNO3 in order to avoid the sample + reagents dispersion on the reflector surface, despite
the silicon coating. It was found that 20 µL of each reagent was a good compromise between the
completeness of the digestion, negligible blank values and minimum dispersion of the sub-sample.
These volumes (20 + 20) have been optimized for a 1.1 mg aliquot. Higher masses of the SRM were
also tried, but it was found that above that amount the material became increasingly difficult to digest,
due to the limited volume of reagents which can be deposited on the reflector without causing
dispersion of the solid. Reagent volumes higher than 20 µL leads to the dispersion of the sample onto
the reflector. As a consequence of this dispersion, the counting rates becomes lower, as could be seen
in Figure 11.
The digestion must be performed in mild conditions in order to minimize the mechanical losses of the
sample due to uncontrolled boiling of the hydrogen peroxide + nitric acid mixture. The optimized
digestion time was 8 minutes. Digestion of the sample, with the subsequent destruction of the matrix
increases the signal-to-background ratio (SBR) and, in consequence, the detection capabilities of the
technique. Table 3 shows the SBR of different elements for crude and digested samples (Bovine liver
standard and whole blood). For most elements, the SBR increases appreciably in the digested sample.
A clear exception is chloride which is partially removed during the digestion. This enhancement is
important for those elements in the low-energy part of the spectrum. Absolute detection limits for the
elements of interest become lower after the digestion procedure [29].
The removal of the organic matrix of the SRM 1577a can be estimated by means of the ratio of the
Co/Compton peak ratio. An enhancement of 40% was found for this ratio in the digested samples
compared to the undigested or crude samples. Recovery values ranged from 34 % (S) to 117 % (Ca),
but in general they were above 88 % (Fe) the elimination of the matrix influences the quality of the
thin layer, being the self-absorption lower, which helps also the increasing of the recovery rate for
potassium and sulphur. Concerning to the elements Ca and Mn, the recovery rates were above 100%
due to the overlapping of the smaller calcium signal with the bigger signal of the element potassium;
in the case of manganase, the effect of the bigger peak of iron seems to be the main reason of the
recovery rate over 100%, since no contribution of the blanks was found. The precision of the
procedure measured as relative standard deviation was bellow 10% in all the cases, when sample
reflectors were rotated 5 times. The precision among independent replicates (n=10) was always less
than 10% RSD for most of the analyzed elements as shown in Table 4.

4.4.- In situ Chemical Modification

Selection of the chemical modifier and modification procedure

123
Contrary to the success with which oxalic acid and ammonium oxalate have been employed in
GFAAS, none of them helped in eliminating the chloride Kα peak, regardless of the heating method.
Additionally, treatment with these reagents rendered solid residues that, even with the naked eye, were
clearly far from the “thin film” condition. Only the addition of ammonium nitrate effectively removed
the interference, and therefore this compound was exclusively used for further study.
The most adequate preparation procedure consisted on drying the sample + modifier aliquots under an
infrared lamp, then heating on a hot plate until the appearance of white vapors. Contamination was
found to be a recurrent problem when using the muffle oven, and its use was thus discarded. By
applying this simple procedure, effective removal of the chloride Kα peak could be assessed,
permitting the determination of calcium and potassium in a model solution at the 1.0 µg ml-1 level and
NaCl 1% w/V matrix. No problems were observed for selenium at this concentration level; a result
that could be in part expected on the basis of its location in the energy spectrum (Kα= 11.21 eV).
Effect of ammonium nitrate as chemical modifier on the analysis of amniotic fluid samples
The procedure briefly discussed in the preceding section was applied to a pooled amniotic fluid
sample. Figure 12 shows the effect of the addition of increasing concentrations of ammonium nitrate
on the relative sensitivity of the chloride Kα peak. The sensitivity decreases progressively until it
reaches a minimum at a modifier amount of 0.4 mg on the reflector surface. Most of the compound is
decomposed and vaporized during the chemical modification procedure, the actual mass remaining on
the carrier is much lower. The latter preserves the quality of the film, thus permitting adequate analysis
of the sample by TRXF. It can also be seen in the same figure the concomitant reduction of the
background intensity in the region comprising the sulfur, chloride and potassium peaks. Evidently, the
low energy part of the spectrum is the most affected by the presence of the chloride ion. A slight
increase in the background intensity is observed at a modifier mass of 0.5 mg. Based on these essays,
0.4 mg of ammonium nitrate was chosen for chemical modification purposes.
Figure 13 shows the spectrum of an amniotic liquid sample with and without the addition of
ammonium nitrate (0.4 mg). Even though the signals for potassium and calcium are clearly
distinguishable in this kind of sample, despite the high intensity of the chloride peak, it can be easily
concluded that problems would arise in those cases where K:Cl and/or the Ca:Cl ratio would be low.
Removal of the interference has a direct impact on the detection limits, particularly in the region of Z<
30, as evidenced in Figure 14.
The addition of the modifier did not affect the quality of the film, permitting quantitation to be carried
out by means of an aqueous standards calibration curve. The results summarized in Table 5 reveal
various interesting aspects. First, eliminating the spectral interference permits a better fitting of the
spectra, which translates in a higher accuracy of the measurements for potassium. Second, the
concentration of calcium in most samples did not differ significantly with and without chemical
modification, with the exception of sample 4. It is deemed that partial volatilization of the analyte was
responsible for the lower recuperation rate. This phenomenon was clearly observed for bromine,
which is a volatile element, and it may be deemed to occur for other elements as well. The latter
though, could be prevented by careful control of the vaporization temperature. Another alternative
would be introducing an element (internal standard) which would thermally stabilize those analytes
susceptible to volatilization losses. The evaluation of such chemical modifiers is currently under
progress in our laboratory. For the elements Fe, Zn and Rb non significant differences were found
between both procedures as deduced from Table 4. Sr was detected only in two samples and after the
chemical modification procedures. This could be a consequence of the improvement in the signal to
background ratio near the Compton peak signal, since strontium signal is closed to this peak.
5.- Conclusions
Direct analysis by TXRF is a suitable method for determination of trace elements in biological
samples as brain tissue, amniotic fluid, serum and urine with adequate analytical quality. The precision
is mainly affected by the element level to detection limit ratio independently of the standardization
method (Compton peak, element as internal standard). The precision and accuracy are similar to those
obtained in the analysis of digested samples. The results obtained using element as internal standard

124
have non significant differences respect to the techniques of FAAS and FIAAS for serum and brain
samples. Amniotic fluid and urine samples have the lack of a high saline content, which affects the
quantification at low element levels. Quantification using aqueous sensitivity curves is possible since
the relative sensitivity values are independent from sample matrix for elements with Z > 19. The
method of Compton peak standardization is suitable for Pt, Cu, Zn, Fe and Se at low levels in serum
and brain slurry samples, due to their constant matrix. The results are accurate, precise and are in
agreement to those obtained using element as internal standard (V or Co), FAAS and FIAAS
techniques. The method overcome the difficulties associated to the use of matched standard in
Compton peak standardization.
The in situ microwave digestion of miligram amounts of a sample directly on the surface of the
reflector surface with a hydrogen peroxide-nitric acid mixture. In order to prevent the sample from
spreading on the reflector surface, the latter must be previously siliconized, the reagents added in a
strict sequence and the digestion conducted in a mild power setting. In comparison to the direct
analysis of the crude sample, the microdigestion increases the signal-to-background ratio, particularly
for low-energy elements, thus improving the detection limits, while providing higher recovery rates
for most of the analytes. An enhancement of the recovery rate is obtained for elements with low Z, as
for example S and K. Certainly, contamination problems and volatilization losses may be more
significant when such low amounts of samples are utilized. Nonetheless, the method may well be
suitable for analysis of samples of which only scarce quantities would be available, e.g., biopsy
tissues, blood samples from small children, etc., if the contamination and volatilization problems are
controlled. Additionally, since the solvent is evaporated during the digestion process, no dilution of
the sample results, thus the detection capabilities are improved.
The applicability of chemical modifiers in TRXF demands new requirements not observed in the
original concept as applied in GFAAS. In this case, besides affecting the component of the system to
which it is addressed (analyte, matrix and/or reflector surface), the chemical modifier must not hamper
the formation of a thin film so that proper total reflection of the incident X-ray beam would occur.
Ammonium nitrate complied with these conditions and effectively remove the chlorides from matrix,
while permitting quantification by means of an aqueous standards calibration curve. Partial
volatilization of some analytes may occur though, so careful control of the temperature is mandatory.
Addition of a modifier which would thermally stabilize those volatile elements may also be an
alternative.

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129
Sample Zn Concentration (µg ml-1)
FIAAS ( %RSD) TXRF
215 0.52 (5%) 0.63±0.07
213 0.390 (2%) 0.49±0.05
212 1.8 (10%) 1.3±0.06
210 0.41(4%) 0.44±0.05
209 0.56 (3%) 0.64±0.04
200 0.73 (2%) 0.94±0.06
207 0.69 (5%) 0.77±0.06
l13 0.120 (7%) 0.25±0.04
l16 0.11 (9%) 0.23±0.05
l18 0.25 (5%) 0.36±0.06
l20 0.17 (8%) 0.30±0.03
l21 0.16 (8%) 0.29±0.04
l22 0.20 (6%) 0.31±0.04
l23 0.13 (8%) 0.26±0.05
l24 0.06 (20%) 0.17±0.03
l25 0.13 (8%) 0.25±0.03
l27 0.09 (12%) 0.19±0.04
l28 0.09 (12%) 0.17±0.04
l29 0.13 (9%) 0.46±0.08
l3 0.08 (15%) 0.18±0.04
l30 0.12 (9%) 0.22±0.03
l35 0.13 (9%) 0.26±0.02
l37 0.14 (7%) 0.23±0.04

Table 1. Zn levels in Amniotic Fluid samples by TXRF and FIAAS

130
Sample Serum Urine

2 0,9 +/- 0,1 4,8 +/- 0,4

4 1,1 +/- 0,1 2,7 +/- 0,3 *3,3 +/- 0,2

3 1,0 +/- 0.1 2,9 +/- 0,2

5 1,1 +/- 0,1 4,6 +/- 0,2

-1
Table 2. Platinum levels (µg mL ) in Serum and Urine

Element Bovine liver Whole Blood

Crude sample Digested Crude sample Digested
S 18.9 33.9 16 18

Cl 15.2 10.8 52.6 18

K 218 284 210 287

Ca 7.0 9.3 11.2 12.7

Mn 3.9 5.5 4.5 4.4

Fe 92 102 823 916

Co 45.9 61.5 29.4 25.6

Cu 133 141 3.10 5.7

Zn 118 134 28.4 34.7

Se 1.8 2.2 DL 3.2

Br 16.9 19.0 24 27

Rb 20.4 25.5 10.7 13.7

Table 3. Signal-to-background ratio for various elements in SRM 1577a and whole blood

131
 Element Found Value µg.g-1 Certified Value µg.g-1 % Recovery

Undigested Digested Undigested Digested

S 2,239 (11.0)* 2,679 (6.0) 7,800** 28.7 34

Cl 3,150 (14) 2,006 (2.8) 2,800** 113 71.6

K 7,487 (17) 9,920 (4.0) 9,960** 75.2 99.6

Ca 120 (16) 140 (5.0) 120 100 117

Mn 9.5 (7.7) 11.4 (8.0) 9.9 96 115

Fe 170 (6.0) 171 (7.3) 194 87.8 88.1

Cu 143 (7.0) 146 (6.0) 158 90.5 92.4

Zn 106 (5.3) 119 (5.8) 123 86 96.7

Br 7.7 (11.0) 8.2 (7.5) 9*** 85.5 91.1

Table 4. Recovery rates for various analytes in the NIST Bovine Liver standard reference material (SRM
1577a). * Values in parenthesis are relative standard deviations for ten independent replics. ** The values
are expressed in percent in the original certificate. *** Non certified value, given as reference in the
certificate. Table taken from [28]

Concentration ( µg ml-1)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

E l. WM WOM WM WOM WM WOM WM WOM WM WOM
K 144 126 135 109 118 98 119 107 124 95
(3) (4) (6) (4) (3) (3) (5) (2) (4) (6)
Ca 78 47 41 53 18,4 29 39 44 32 42
(8) (4) (1) 3 (0,5) (2) (3) (2) (1) (3)
Fe 0.4 ND 1.06 1.0 0.50 0.45 0.40 0.4 0.58 0.60
(0.1) (0.07) (0.1) (0.06) (0.08) (0.08) (0.1) (0.06) (0.05)
Zn 0.18 0.16 0.45 0.50 0.26 0.36 0.24 0.29 0.45 0.6
(0.01) (0.06) (0.03) (0.03) (0.03) (0.03) (0.01) (0.06) (0.03) (0.1)
Br 3.20 6.3 2.4 4.02 1.69 3.86 2.28 4.43 1.91 4.13
(0.08) (0.2) (0.1) (0.08) (0.03) (0.07) (0.09) (0.06) (0.08) (0.02)
Sr 0.042 Nd Nd Nd Nd Nd Nd Nd 0.033 Nd
(0.004) (0.006)
Rb 0.13 Nd 0.09 0.075 0.08 0.135 0.087 0.10 0.12 0.11
(0.01) (0.01) (0.002) (0.03) (0.007) (0.007) (0.01) (0.02) (0.02)
Table 5. Comparative results of the determination of Fe, Zn, Sr, Se and Br in amniotic fluid samples with
(WM) and without (WOM) chemical modification

132
 200

Compton peak

150
Intensity (counts/channel)

Br-Kα

100

50 Co (Std.I)
Sn-L Pb-L
Pt-L

-50
2 4 6 8 10 12 14 16 18 20

Energy (keV)

Figure 1. Spectrum of spiked serum sample for L-lines calibration

300 Compton peak

Se-Kα
250
Intensity (counts/channel)

200

150

Co-Kα
100

50 V-Kα

4 8 12 16 20

Energy (keV)

Figure 2. Spectrum of spiked brain slurry sample for K-lines calibration

133
 3.5

3.0

2.5

2.0
Sensitivity/V

1.5

1.0

0.5

0.0
22 24 26 28 30 32 34 36

Atomic Number

.
Figure 3. Sensitivity curves in urine, water and serum matrices using vanadium as internal standard.
Serum (—), water( ) and urine(●)

2,5

2
Concentration ( g mL )
-1

1,5

0,5

0
Zn Cu

Figure 4. Zn and Cu concentration in brain slurries determined by direct TXRF analysis compared to
TXRF of digested sample and FIAAS. TXRF-Vanadium as internal standard (□), TXRF – cobalt internal
standard (■), TXRF-digested sample (■) and FIAAS(■).

134
 2.4
Concentration (µg mL )
2.0
-1

1.6

1.2

0.8

0.4

0.0
18 20 22 24 26 28 30 32 34 36
Atomic Number

Figure 5. Detection Limits in Amniotic Fluid (■), Amniotic fluid 1:1( ), serum ( ), Water (●) and

1000

100
Concentration (8 g mL )
-1

K
10 Ca

Fe
1 Cu

Zn
0,1
Se

Br
0,01
0 2 4 6 8 10 12

Label of individual
urine(□)
Figure 6. Levels of K, Ca, Fe, Cu, Zn, Se and Br in urine samples by direct TXRF analysis

135
 10
Intensity (counts/channel)

50
Co (I.S) Br
Pt

Zn
0
4 8 12 16 20
Energy (keV)

Figure 7. Spectrum of typical urine sample from a cancer patient 24 hours after treatment with Pt drugs

0.008

8
Sensitivity/V ; Sensitivity/Compton L- Lines

Sensitivity/Compton K- Lines

0.006
6

0.004
4

0.002
2

0 0.000

20 30 40

Atomic Number

Figure 8. Sensitivity curves for TXRF calibration. K-lines in Aqueous standard and Vanadium as Internal
standard(□
□), K-lines in serum matrix and Compton peak as internal standard (●
●), K-lines in brain slurry
and Compton peak as internal standard( ), L-lines in serum and Compton peak as internal standard(■)

136
 2.5

Concentration (µ g mL-1)
2

1.5

0.5

0
Fe Cu Zn Se Zn Cu Pt
serum serum serum serum brain brain serum

Figure 9. Comparative results using element as internal standard (□

□) and Compton peak as internal
standard (■)

2.5

2
Concentration ( g mL )
-1

1.5

0.5

0
Fe serum Cu serum Zn serum Cu brain Zn brain

Figure 10. Comparative results using TXRF and Compton peak as internal standard(■)and FAAS
technique (□
□)

137
 16000

14000
Intensity (counts/channel) 12000

10000

8000

6000

4000

2000

18 20 22 24 26 28 30
Atomic Number

Figure 11. Effect of the reagents volume on the TXRF signal for the in situ microwave digestion of 1.10 mg
of the Bovine liver standard sample 1577a. Non digested sample(■), 20 mL HNO3 - 20 mL H2O2 (▲) and
40 mL HNO3 – 40 mL H2O2 (●
●)

1.6
6
1.4
5
1.2
Σ (BS, Cl, K) / I IS

4
1.0
ICl /I IS

3
0.8
2
0.6
1
0.4
0
0.2
-1 0 1 2 3 4 5 6
4 --1
Mass of NH4NO3 (10 µg ml )

Figure 12. Optimization of the mass of ammonium nitrate for elimination of the chloride Kα peak in a
pooled amniotic fluid sample. Left scale intensity of Cl signal to internal standard ratio: ICl/I IS ( ); right
scale sumatory of background area of S, Cl and K to Internal standard ratio: Σ (BS,Cl,K )/ I IS ( )

138
 3000 Mo
Cl
2500
Intensity (counts/channel)
2000

1500
Ni
1000 K (Int. Std)
Ca
Br
500

2 4 6 8 10 12 14 16 18
Energy (keV)

Figure 13. Spectrum of an amniotic fluid sample with (dotted line) and without (continuous line)
ammonium nitrate (0.4 mg) as chemical modifier. Ni was used as Internal Standard (IS)

0.35

0.30
(µ g. ml -1)

0.25

0.20

0.15
Detection Limit

0.10

0.05

0.00

22 24 26 28 30 32 34 36 38 40
Atomic Number

Figure 14. Detection limits in an amniotic fluid sample as a function of the atomic number for different
masses of ammonium nitrate chemical modifier: no modifier (¡); 0.1 mg (c); 0.2 mg (•); 0.3 mg (c); 0.4
mg (d); 0.5 mg ( )

Lué-Merú Marcó P.
Universidad Centro Occidental Lisandro Alvarado
Decanato de Agronomía. Núcleo Tarabana. Dpto. Química y Suelos
Tarabana. Cabudare. EDo. Lara. Venezuela
luemerumarco@yahoo.es

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 7
X-Ray Spectrometry for Analysis of
Atmospheric Particulate Matter:
Detection Limits Versus Legal Levels

R. Van Grieken, Y. Makarovska, K. Van Meel and

A. Worobiec

Introduction
For multi-elemental analysis of aerosols deposited on filters, X-ray emission spectrometry (XRS)
analysis has since long been a powerful alternative method to other analytical techniques, as e.g.
inductively-coupled plasma mass spectrometry (ICP-MS). Numerous research institutes and
environmental monitoring agencies apply X-ray fluorescence (XRF) to analyze aerosols collected in
different locations in order to estimate the level of air pollution, mostly with respect to heavy metals.
In fact, the first conference on aerosol analysis by XRS took place as long ago as April 1972, in
Seattle, WA. Since then, about 60 articles have been published annually, continuously over the last
three decades, on XRS for aerosol analysis (Injuk and Van Grieken, 2003). This makes aerosol
analysis one of the major “classical” applications of XRS. The reason is very simple: aerosol loaded
filters are ideal targets for XRS, since they are thin and homogenously loaded, and often the aerosol
deposit is so thin that hardly any X-ray absorption correction is needed. Filters constitute a thin clean
substrate on which the particulate matter is deposited uniformly. They can be analyzed directly; no
sample pre-treatment is required. In this way, systematic errors arising from the sample preparation
can be avoided. Quantitative measurements of aerosols require the analysis of thin film standards
which have to be uniform and very thin.
In the early days, XRF was mostly used to screen heavy metals in aerosols. This was important since
heavy metals are a health hazard, and it can be assumed that some 50% of the human uptakes occurs

140
via the air. We inhale directly ca. 1/3 of our heavy metal intake, and about 1/3 come via both food and
water ingestion. But the latter two vectors are also related to heavy metals in the air, since vegetables
and drinking water reservoirs are prone to atmospheric deposition of heavy metals. There have been
three other aspects which have made aerosols of immense renewed interest in the last decade or so: (1)
they significantly affect the Global Climate by direct sunlight scattering and enhanced cloud formation
and may or may not compensate for the well-known effect of greenhouse gases, (2) the main health
hazard of air pollution is no longer considered to be neither sulfur dioxide (since this component has
decreased so much in the industrialized world recently) nor ozone, but fine particulate matter (e.g.
PM2.5, with and aerodynamic diameter below 2.5 µm) which causes much more significant morbidity
and mortality via lung and heart problems, and (3) the main threat to cultural heritage items is no
longer sulfur dioxide and acid rain but the deposition of atmospheric particles on indoor works of art.
Practically all types of XRS have, of course, been used for analysis of aerosols; this includes tube-
excited and radioisotope-excited XRF, total-reflection XRF (TXRF), particle-induced X-ray emission
analysis (PIXE), and, mostly for single particle analysis, electron-probe X-ray microanalysis
(EPXMA), micro-XRF and synchrotron-induced XRF (SXRF).
While most routine XRF spectrometers today still operate in the wavelength-dispersive mode
(WDXRF), ca. 15,000 worldwide, the number of stand-alone energy-dispersive (EDXRF)
spectrometers is significant as well, around 4,000 now. The common EDXRF mode provides
simultaneous determination of different elements, but often offers insufficient sensitivity and accuracy
for low-Z elements. A typical EDXRF spectrum obtained with a conventional instrument tube-excited
EDXRF instrument is shown in Figure 1.
Intensity, a.u.

0 10 20
Energy, kV

Figure 1. Typical spectrum of an aerosol loaded filter, obtained with a conventional EDXRF unit (Tracor-
5000)

As the part of the sample actually analyzed in XRS has typically a surface of only few cm2, it must be
representative for the entire sample. Therefore, a critical step is in the selection of an appropriate filter
material. Teflon and polycarbonate (Nuclepore) filters are ideal because of their high purity and
because they are surface collectors. Whatman-41 cellulose filters have been used widely because of
their low cost and low air-flow resistance, but they are rather thick (namely around 10 mg/cm2 versus
1.1 mg/cm2 for Nuclepore), which leads to more X-ray scatter and higher detection limits (DLs), and
they partially collect particulate matter within their depth, so that X-ray absorption corrections become
more complicated. Glass fiber filters should be avoided in any case due to their high inorganic

141
impurity. High-purity (but expensive) quartz fiber filters have more recently been proposed. It is rather
remarkable that, although XRS for aerosol analysis has been around for more than three decades, no
very systematic evaluation and comparison of filter materials has been published in the open literature;
rather, each user has his own preference, based on own experience and on the grey literature.
Because of the immense loss of intensity in the crystal dispersion based detection system, WDXRF
implies very much higher excitation intensities than EDXRF. This often means that common cheap
cellulose or thin polycarbonate aerosol filters are destroyed rapidly in the exciting beam of WDXRF;
typically, this will occur after a few minutes or irradiation, and no prolonged measurements to
improve the sensitivity, and no repeated analyses, are normally possible. In spite of this, WDXRF has
been the XRS method of choice in several European environmental agencies, in view of its higher
accuracy and sensitivity for low-Z elements. This may change to some extent in the near future, as will
be discussed below.

Will There Soon Be a Sensitivity Problem for WDXRF Analysis of Heavy Metals
In Aerosols?
In the last two decades, the emission rates and hence also the atmospheric concentration levels and
deposition rates of heavy metals have dramatically decreased in most countries. While e.g. the
emission of Pb over Europe was in total 92,000 tons/year in 1980, it decreased to 89, 37, 18 and 12
kton/y in 1985, 1990, 1995 and 2000, respectively. E.g. the atmospheric Cd concentrations over
Europe were in the range 0.1-0.7 ng/m³ in 1980; they decreased to 0.07-0.25 ng/m³ in 2000.
On the other hand, the legally acceptable limits have evolved too. What are the bodies that define the
legal maximum concentrations?
-At the international level, the World Health Organization (WHO) in Geneva has issued Guide Values
for Air Quality in 2000. These values are not enforced but are guidelines indeed.
-At the European level, there is, since 1996, an Air Quality Framework (European legislation
96/62/EC), to be specified be so-called Daughter Directives. The first Daughter Directive was issued
in 1996 (European legislation 1999/30/EC) and concerned Pb, particulate matter, SO2 and NO2-NOx).
The Fourth Daughter Directive appeared in 2004, and concerned poly-aromatic hydrocarbons and the
heavy metals As, Cd, Ni and Hg. The latter limits will be strictly enforced from 2010 on.
-Thirdly, there are limits at the national or regional level. E.g. for Flanders, the northern region of
Belgium, the legislation on heavy metals is included in the so-called VLAREM Title IIbis norms. In
the USA, it is the Environmental Protection Agency that deals with the environmental legislation,
namely the national ambient air quality standards (NAAQS).
When one inspects these legislations for the most stringent limit, the data represented in Table 1 are
obtained.

Element Most stringent level (in ng/m³) Legislation

V 1000 WHO

Mn 150 WHO

Ni 0.38 WHO (for exposed persons)

10 Future EU norm

As 1.5 WHO

142
 4 Future EU norm

Cd 5 WHO; future EU norm

Hg 1000 WHO

Pb 500 WHO; present EU norm

1500 USA NAAQS

Table 1. Most stringent limits for atmospheric heavy metal concentrations (in ng/m³)
In the USA, legal monitoring is done by EDXRF, using dichotomous sampling, i.e. sampling which
results in aerosol filters containing the inhalable and non-inhalable fractions separately. In e.g.
Belgium, France and Germany, atomic absorption spectrometry and inductively-coupled plasma
atomic emission spectrometry are legally recommended, but XRF may be applied after it has been
calibrated versus the former techniques. In Belgium, some 130 aerosols filters have been measured
daily for two decades by WDXRF for official heavy metal monitoring purposes.
Are the detection limits of the common WDXRF technique suitable for attaining the present and future
most stringent maximum concentration levels? One problem is that the detection limits for a technique
are obviously expressed in ng/cm² of the loaded filters, while the legislation is in ng/m³. Conversion
between these values is not always straightforward and depends obviously on many factors:
*the length of the sampling period
*the pump power and the air flow rate
*the size of the filter
*the filter type. As mentioned above, although a very systematic study has never been published to our
knowledge, many recommendations are available, e.g.:
(1) glass fiber filters can never be used because of their significant inorganic blank!
(2) cellulose fiber filters allow a high air flow rate but the aerosol is captured partially within the filters
which may lead to complicated X-ray matrix effect corrections, the filters are rather thick (ca. 10
mg/cm²) leading to a relatively high X-ray background and the filters are easily damaged by the X-ray
beam in WD-XRF
(3) quartz fiber filters allow a high air flow rate but the collection also occurs within the filter and they
are rather thick and expensive
(4) polycarbonate membranes, like Nuclepore filters with controlled pore-size, are thin (1 mg/cm²) and
collect at their surface only but the pressure drop over the filter is high and hence the air flow is low,
and damage occurs quickly in a WDXRF instrument
(5) cellulose nitrate membranes have intermediate characteristic but they are also damaged in the
WDXRF beam.
Let us transform the most stringent maximum airborne levels as shown in Table I, for the four heavy
metals that will soon be implied and controlled in the EU legislation, via the experimental sampling
conditions as used by the Flemish Environmental Agency (VMM), in which 55 m³ are pumped in 24 h
through a cellulose nitrate filter with and effective area of 12.8 cm². In this way, the data in Table I can
then be transformed to the ones given in the first numerical column in Table 2. The realistic DLs that
have been discussed by Vanhoof et al. (2003), implying a typical 2 min counting per element in a
WDXRF setup with a Rh tube, are given in the last column of Table 2.

143
Element DLs required to meet the most stringent Typical WDXRF DLs (in
legislation (in ng/cm²), based on the sampling ng/cm²) according to Vanhoof
strategy of VMM et al. (2003)

Ni 1.66 (WHO for exposed persons) 11

44 (future EU norm)

As 6.6 (WHO) 55
17 (future EU norm)

Cd 22 (WHO; future EU norm) 66

Pb 2200 (WHO; future EU norm) 44

6560 (USA)

Table2. Required analytical detection limits (DLs) to reach the most stringent legal maxima in Table I, as
compared to the DLs of a typical aerosol collection strategy and a WDXRF setup

From Table 2, it is obvious that for As, Cd and partially for Ni, the typical DLs for WDXRF will soon
not suffice to keep up with the reduced maximum concentration levels implemented by the
forthcoming legislation.

Will the New High-Energy Polarized-Beam EDXRF Be Capable of Offering

Sufficient Sensitivity?
Many years ago, Kaufmann et al. (1974) and Standzenieks et al. (1979) were among the first to
investigate the use of a three-dimensional optical geometry with polarized X-ray beam for EDXRF. A
combination of a secondary target and a three-dimensional polarizing optical geometry drastically
reduces the scattered X-ray spectrum from the tube and hence it causes a significant decrease of the
detection limits. Application of a high-energy X-ray source is also promising due to the possibility to
use K-lines for heavy element analysis, if a detector with advantageous sensitivity at high energy is
used, like a Ge-detector. Compared to L-lines, K-lines undergo much less spectral interference and
matrix effects. Only few publications dedicated to the use of polarized (P) high-energy (HE)-EDXRF
conventional laboratory spectrometers have been found in the literature.
The decrease of DLs, particularly for heavy elements, is very promising for the application of such
spectrometers for analysis of aerosols. However, hitherto these instruments were not widely used for
analysis of such samples. Viksna et al. (2004) successfully applied a three-dimensional home-built
EDXRF spectrometer equipped with a conventional Ag X-ray tube to analyze the elemental size
distribution and environmental mobility of airborne particles in Riga, Latvia. El-Tahir et al. (2005)
also employed this spectrometer with the same measurement conditions to examine airborne particles
in Khartoum, Sudan. Bennet et al. (2005) investigated particulate air pollution in Dar-Es-Salaam,
Tanzania. Commercial polarized beam instruments have, however, only become available recently,
after 2000, from both Spectro and PANalytical..
We will discuss here the possible use of the commercial HE-P-EDXRF spectrometer, Epsilon 5
(PANalytical, Almelo, The Netherlands), for quantitative analysis and legal monitoring of aerosol
samples deposited on filters.
This instrument has a three-dimensional polarizing geometry with 13 secondary targets (W, CeO2, CsI,
Ag, Mo, Zr, KBr, Ge, Co, Fe, Ti, CaF2, Al) and two Barkla targets (B4C and Al2O3). The Gd-anode X-
ray tube can be operated at an accelerating voltage of 25-100 kV and a current of 0.5-24 mA
(maximum power 600 W). The X-ray characteristic radiation is detected by a Ge-detector (PAN 32)

144
with a FWHM resolution of 165 eV or better, at 5.9 keV. There is a possibility to use two mediums for
the measurement: vacuum and helium. As was mentioned by Nakai (2004), a combination of
secondary targets and a three-dimensional polarizing optical geometry greatly reduces the scattered X-
ray tube spectrum. The observed background was very low. Therefore, for the optimization of the
measurement conditions, only the absolute values of the characteristic peak intensities were used. The
combination (target, accelerating voltage and current) which ensured the highest intensity of the peak
was chosen to be the best condition. The target giving rise to the highest peak can be considered to be
the best. The best conditions of the measurement of X-ray characteristic radiation chosen finally for
each analyte are presented in Table 3.

Element Optimal secondary Tube voltage used

target (in kV)

Al to Ca Ti 35

Ti to V Fe 50

Cr to Zn Ge 100

Se, Pb Zr 100

Sr Mo 100

As KBr 100

Cd CsI 100

Sb CeO2 100

Table 3. Optimal analysis conditions of HE-P-EDXRF for aerosol loaded filters

The analytes were combined in 8 groups according to the chosen measurement conditions. Availability
of many secondary targets allows also solving the important problem of the overlapping of the
characteristic lines. For example, the KBr secondary target is the optimal one to excite the As Kα line.
Since the energy of Br Kα line is above the As K-edge and below the Pb L-edge, the excitation of the
Pb Lα line is significantly reduced. Therefore, the overlapping of As Kα and Pb Lα lines is mostly
overcome and the strong Kα line of As instead of the weaker Kβ can be used. Pb, on the other hand, is
analyzed using the Zr-target (see Table III). The software offers some overlap-correction possibilities,
allowing the use of the Pb Lα line for analysis when As is also present in the sample.
To prepare standard filters, an aerosol generation system was used, by nebulizing an aqueous standard
solution. After drying these aerosols, they were collected on blank filters (Nuclepore filters of 0.4 µm
pore-size and 47 mm diameter). These samples were used later as primary standards.
The accuracy of the applied method and of the obtained calibration curves was checked by the
measurement of a standard reference material from NIST (2783 Air Particulate on Filter Media). The
obtained results were in a good agreement with the certified reference values.
The precision was usually better than 1%, which is very satisfactory for EDXRF.
DLs were calculated according to the usual criterion of three times the square root of the background.
To estimate this, a NIST 2783 blank filter was measured 10 times according to the best conditions

145
chosen for such analysis and the obtained calibration curves. The results are presented in Table IV for
a 300 s counting time.

DLs of HE-P-EDXRF for 300 s counting (in DLs required to meet the most stringent
Element
ng/cm²) legislation (in ng/cm²), as in Table I

Si 30

K 5

Ca 4

Ti 3

V 2

Fe 8

Se 9

Sr 3

Cr 5

1.66 (WHO for exposed persons)

Ni 3 44 (future EU norm)

Mn 4

Cu 3

Zn 4

Cd 10 22 (WHO; future EU norm)

Sb 20

2200 (WHO; future EU norm)

Pb 8 6560 (USA)

6.6 (WHO)
As 3 17 (future EU norm)

S 8

Al 32

Cl 6

Table 4: DLs with the new HE-P-EDXRF instrument, as compared to the most stringent legal limits

146
Of course, the detection limits can further be improved according to 1/t1/2, by counting longer. An
acquisition time of 300 s was considered as a reasonable compromise between low DL and time of
analysis. It should be also stressed that the application of any of the primary beam filters installed in
the spectrometer did not really improve the DLs. The filters are applied to cut a certain part of the tube
radiation in order to avoid the excitation of a part of the spectrum not being of interest at the moment.
Such approach leads to the decrease of the background caused by scattered radiation. However, the
background in this application appears to be sufficiently low and hence the use of primary beam filters
is not beneficially.
When comparing the DLs obtained in this investigation (for an acquisition time of 300 s) with data
obtained by WDXRF (Vanhoof et al., 2003), it is clear that the results obtained by the HE-P-EDXRF
spectrometer Epsilon-5 are significantly more sensitive. It demonstrates that the application of HE-P-
EDXRF instrument for analysis of aerosol filters is very promising.
In fact, there is no risk that monitoring the environmental atmospheric concentrations of heavy metals
relative to legal standards by HE-P-EDXRF may be hampered by insufficient sensitivities, as would be
the case for WDXRF. And, in fact, VMM has already decided to do the legal monitoring of heavy
metals in the air in Flanders from now on by HE-P-EDXRF, instead of the WDXRF that was applied
for 2-3 decades in Flanders and Belgium.

References
C. Bennet, P. Jonsson and E. Selin Lindgren (2005), Concentrations and sources of trace elements on
particulate air pollution, Dar Es Salaam, Tanzania, studied by EDXRF, X-Ray Spectrom. 34, 1-6.
H.M. El-Tahir. E. Selin Lindgren and F.I. Habbani (2005). Elemental characterization of airborne
particles in Khartoum, Sudan, X-Ray Spectrom. 34, 144-152.
J. Injuk and R. Van Grieken (2003) Literature trends in x-ray emission spectrometry in the period
1990-2000 - a review, X-Ray Spectrometry, 32, 5-39
D.L. Kaufman and Camp D.C. (1974) Polarized radiation for X-ray fluorescence analysis, Adv. X-Ray
Anal., 18, 247-257
I. Nakai (2004) “High-energy X-ray Fluorescence” in X-Ray Spectrometry: Recent Technological
Advances, K. Tsuji, J. Injuk and R. Van Grieken, Eds. (Wiley, Chichester, England), 1st ed., Chap. 5.5,
p. 355
P. Standzenieks and E. Selin (1979) Background reduction of X-ray fluorescence spectra in a
secondary target energy dispersive spectrometer. Nucl. Instr. Meth. 165, 63-65.
C. Vanhoof., H. Chen, P. Berghmans, V. Corthouts, N. De Brucker and K. Tirez. (2003) Risk
assessment study of heavy metals in ambient air by WD-XRF spectrometry using aerosol-generated
filter standards. X-Ray Spectrom. 32, 129-138.
A. Viksna, E.Selin Lindgren, P. Standzenieks and J. Jacobsson (2004). EDXRF and TXRF analysis of
elemental size distributions and environmental mobility of airborne particles in the city of Riga, Latvia.
X-Ray Spectrom. 33, 414-420.

R. Van Grieken, Y. Makarovska, K. Van Meel and, A. Worobiec

Department of Chemistry, University of Antwerp
BE-2610 Antwerp, Belgium
rene.vangrieken@ua.ac.be

147
 8
In-Situ XRF Analysis

Andrzej Markowicz

Abstract
The paper presents major aspects pertinent to in-situ applications of XRF technique including
excitation sources and typical measurement geometries, parameters describing in-situ analysis, major
interfering effects and associated correction procedures and examples of selected applications.

1.- Introduction
X-ray fluorescence spectrometry is perhaps the first spectroscopic technique which can be successfully
applied in the field and in industrial environment for in-situ analysis of various materials. Modern,
high resolution, portable XRF analyser brings to the field site not only an excellent performance often
matching that iof the laboratory instrument, but also unsurpassed savings in time and labour. Field-
portable X-ray fluorescence (FPXRF) is an example of a well-balanced compromise between
portability, ruggedness, reliability and analytical performance. Simplicity, speed of operation and
flexible requirements for sample preparation are important features of FPXRF techniques. Major
advantages of FPXRF over laboratory-based analysis include availability of immediate analytical
results (important, e.g., for interactive study of contaminated sites), and non-destructive character of
analysis of objects which cannot be sampled or removed to the laboratory for analysis (e.g., works of
art, archaeological samples, museum objects).

2. Excitation Sources and Measurement Geometries

Excitation sources applied in portable XRF analysers include both radioisotopes and low-power X-ray
tubes. Characteristics of most commonly applied sources are in Table. 1.

148
Radioisotope Half live (years) X- or γ-ray energy (keV) Photons per disintegration
55
Fe 2.7 MnK X-rays (5.9; 6.5) 0.28
109
Cd 1.3 AgK X-rays (22; 25) 1.02
γ-rays (88) 0.04
γ-rays (59.5)
241
Am 433 0.36

Table 1. Properties of radioisotope sources applied in portable XRF analysers

Typical geometries applied in radioisotope-excited portable XRF spectrometers include: (i) annular
source geometry, (b) central source geometry, and (c) side source geometry; each has its own specific
advantages and limitations. In portable X-ray tube based XRF analysers three typical excitation
geometries can be used: (a) direct excitation, (b) secondary target excitation, and (c) transmission
geometry.
A wide range of detectors can currently be applied in portable XRF analysers such as scintillation and
proportional detectors (in low-resolution systems) and solid state detectors (in high-resolution
systems). The last group of detectors include liquid nitrogen cooled detectors (Si/Li, HpGe) and no
liquid nitrogen cooled detectors (Si-PIN, CdTe, CdZnTe, silicon drift detectors (SDD)).

3.- Portable XRF Spectrometers

Portable XRF systems can be constructed as two piece set-ups including excitation source, detector,
preamplifier and sample holder in a hand-held probe connected with a second unit containing
electronics and power supplies. There are also one piece hand-held systems where all components are
fully integrated.
Examples of portable XRF analysers constructed by the IAEA Laboratories at Seibersdorf are
presented on Figures 1-3.

Figure1. Portable XRF unit based on radioisotope excitation and liquid nitrogen cooled Si(Li) (or HpGe)
detector

149
 Figure2. Portable XRF unit based on radioisotope excitation and thermoelectrically cooled Si-PIN
detector

Figure 3.Portable XRF spectrometer based on low-power X-ray tube excitation and thermoelectrically
cooled Si-PIN detector

150
4.- Parameters Describing in-situ Analysis
One of the major parameters which should be considered for any XRF application (particularly
important for in-situ XRF analysis of unprepared and often heterogeneous materials) is the critical
penetration depth dcrit defined as the sample thickness from which 99% of a fluorescence signal
originates

dcrit = mthick/ρ = 4.61/(ρµtot) (1)

µtot = µ(E0)cscψ1 + µ(Ei)cscψ2 (2)

where ρ is density of the analysed material; µ(E0) and µ(Ei) are total mass attenuation coefficients of
the primary and characteristic radiation in the whole sample, respectively; ψ1 and ψ2 are the incident
and takeoff angles, respectively.
Critical penetration depths (called also information mass) for selected elements in different matrices
characterised by average atomic numbers are given in Table 2.

Element dcrit (µm) for average atomic number 12.5 dcrit (µm) for average atomic number 15
P 12 79
Ca 50 32
Zn 540 340
Pb 930 570

Table 2. Critical penetration depths for selected elements in materials of different matrices (109Cd
excitation)

One can easily calculate that a layer of 50 % of dcrit contributes 90 % of the fluorescence signal, and a
layer of 15 % of dcrit contributes 15 % of the fluorescence signal; the figures are essential in the
interpretation of the results obtained for the heterogeneous or contaminated materials, and materials of
irregular surface.
Precision is defined as a degree of mutual agreement between the results of the replicate
measurements and can be assessed by analysing a sample containing the elements to be determined.
Accuracy is defined by the difference between the experimental result obtained by using the analytical
technique and the reference (or true) value. The parameter can be assessed by analysis of standard
reference materials or analysis of confirmatory samples by using a reference method.
Blank Samples are extremely important for in-situ XRF analysis. One has to use an instrumental blank
to verify contamination introduced by the spectrometer itself and a method blank to monitor the
laboratory-introduced contamination or interferences.
Total Uncertainty of the XRF measurements includes the individual sources of uncertainties
associated to various stages of the analytical process including sample representation, sample
collection and handling, sample preparation, measurement and quantification.

5.- Interfering Effects and Associated Corrections

151
In in-situ XRF analysis one has to expect the influence of a number of interfering effect resulting from
the analysis of unprepared samples. The effects include:(1) physical matrix effects, (2) chemical
matrix effects, and (3) spectral interferences.

5.1.- Physical Matrix Effects

The effects are associated to variations in morphology or physical nature of the samples.
Particle Size Effects result from the dependence of the intensity of the characteristic radiation on the
shape and size (or size distribution) of the particles in a granular sample. If a sample contains fine
particles, the XRF gives a higher concentration that for a sample containing large grains. The only
practical method to reduce the errors introduced by the particle size effects is to grind the material to
be analysed and sieve it to a uniform and small particle size fraction (particularly important for
determination of the light elements).
Surface Irregularity Effects are critical in the analysis of geological and archaeological samples. They
result in a systematic decrease of the intensity of the characteristic radiation from an irregularly shaped
sample compared to a flat sample of the same composition. The effects can be described theoretically
by using the so-called unevenness factor. Most correction methods are based on the Compton and
Rayleigh scattered radiation. One can also use a special calibration procedure in which the relative
instrumental calibration factors (relative to a chosen reference element) are determined experimentally
based on a set of calibration samples.
Mineralogy Effects are related to the dependence of the intensity of the characteristic X-rays on size,
distribution and position of minerals inside the excited volume. In the context of these effects one has
also to consider the excitation-detection efficiency of the portable XRF analyser. The effects can be
quantified by making replicate measurements for randomly selected non-overlapping points on
representative samples; the results can be used to assess minimum number of the replicate
measurements in order to achieve the required precision of the analysis.
Moisture Effects may be a major source of uncertainty of the analytical results of surface soil or
sediments. In order to correct the results and present them on a dry sample basis, an independent
determination of moisture content is needed. Correction for the moisture effects can also be made by
using the measurements of the intensities of the primary scattered radiation at two different energies.

5.2.- Chemical Matrix Effects

These effects (called also matrix effects) manifest in dependence of the intensity of the characteristic
X-rays on the overall composition of the analysed sample. The so-called interfering elements can
result in absorption or enhancement of the characteristic X-rays of the element to be determined. There
is a wide range of the matrix correction methods including methods based on scattered primary
radiation and backscatter fundamental parameter (BFP) methods. The methods based on scattered
primary radiation are relatively simple and are particularly suitable for determination of one (or a few)
elements. The best results are obtained for the elements with the highest atomic numbers (compared to
the interfering elements present in the samples). The BFP methods make use of the Compton and
Rayleigh scattered radiation to characterise the dark matrix consisting of light elements whose
characteristic X-rays are not present in the recorded spectrum. The BFP methods are more versatile
and particularly suitable for determination of a larger number of elements present in complex samples.

6.- Selected Applications

The portable XRF spectrometer based on radioisotope excitation and thermoelectrically cooled Si-PIN
detector (see Figure 2) was applied for in-situ analysis of unprepared soil; BFP method was used for
quantification of in-situ measurements. Next three sub-samples of soil material was taken from non-
everlapping points within the examined area for laboratory analysis which included including

152
homogenisation and sieving. The agreement of the results for in-situ and laboratory measurements was
satisfactory except for Zr. Most likely homogenisation process released Zr from “hidden” grains
(which did not contribute to the fluorescent signal recorded during in-situ measurement) which
resulted in much higher concentration of Zr obtained by using the laboratory method. The fact
confirmed that quantitative results of in-situ measurements must be considered/interpreted with
maximum care.
The portable XRF spectrometer based on low-power X-ray tube excitation and thermoelectrically
cooled Si-PIN detector (see Figure 3) was applied for study of cultural heritage artefacts in the
Museum of Fine Arts in Vienna, Austria. They included: (1) paintings - to support fast identification
of retouching materials and pigments in restoration and study of provenance, and (2) sculptures and
alloys - to support study of their provenance.

A few selected publications for further reading are below:

R. Cesareo, X-ray Physics: Interaction with Matter, Production, Detection, La Rivisita del Nuovo
Cimento della Societa Italiana di Fisica, Bologna, 2000
R.E. Van Grieken and A.A. Markowicz, Eds, Handbook of X-ray Spectrometry – Second Edition,
Revised and Expanded, Marcel Dekker, New York – Basel, 2002
R. Tertian and F. Claisse, Principles of Quantitative X-ray Fluorescence Analysis, Heyden, London,
1982
K. Tsuji, J. Injuk and R. Van Grieken, X-ray Spectrometry: Recent Technological Advances, John
Wiley & Sons, Ltd, Chichester, England, 2004

Andrzej Markowicz
Instrumentation Unit
IEAE Laboratories A-2444
Siebersdorf, Austria
A.Markowicz@iaea.org

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 9
Particle Induced X-Ray
Emission (PIXE)

Javier Miranda

Abstract
One of the most successful X-ray analytical techniques, which is based on particle accelerators, is the
method known as Particle Induced X-ray Emission (PIXE). (Johansson and Campbell, 1988;
Johansson et al., 1995; Maenhaut and Malmqvist, 2002). The main reasons for this are its
multielemental analysis capabilities, good sensitivity and fast results. In this chapter, there is a brief
description of the most important characteristics, as well as examples of applications.

1.- Basic Physical Processes

The basic physical phenomenon for PIXE is the ionization of atomic inner shells by the impact of an
energetic positive ion, followed by the de-excitation of the atom by the decay of an upper-shell
electron to fill the vacancy, and the subsequent emission of an X-ray photon, in order to eliminate the
excess energy in the atom (Figure 1). In this case, there are also other processes, like the Auger
electron emission or the radiative Auger process (simultaneous emission of a photon and an electron).
The energy of the X-ray photon is characteristic of the emitting atom, so the latter can be identified
after measurements of the photon energy.

154
 X-ray photon Electron to
Expelled electron
fill vacancy

Incident ion

Figure 1. Schematics of the basic physical process involved in PIXE

Due to the existence of discrete energy levels in the atom, the electron expelled in the process describe
above may come from different atomic shells, as well as the electron filling the vacancy. There are
well-determined groups of X-ray lines, with a name according to the shell of the ejected electron.
Thus, a line created by a vacancy in the K-shell (principal quantum number n = 1) is called a K line, a
vacancy in the L-shell (principal quantum number n = 1) gives raise to an L-line, and so on.
Now, the electron filling the vacancy may have an origin form many other energy levels in the atom,
limited only by the quantum selection rules for electronic transitions. This fact makes possible to have
a number of K, L, M or other lines, which must also be identified. Therefore, the most intense line in
the K group is called K , ant the next one is the K . Each one of these lines may also be composed of
a certain number of lines, which are recognized by the subscripts 1, 2, 3, etc., so there are K 1, K 2,
K 1, K 2, … lines. Figure 2 displays the most important lines for PIXE analysis. This nomenclature is
known as the Siegbahn notation, while there is a more recent notation developed by the International
Union of Pure and Applied Chemistry (IUPAC) (Markowicz, 2002).
5

N
{ 4
3
2
1

M
{ 4
3
2
1

L
{ 3
2
1
L L L L L L L L L L
l α α β η β γ β β γ
2 1 2 1 1 4 3 3

K
K K K
α α β
1 2 1

Figure 2. Atomic energy levels and electronic transitions giving rise to characteristic X-rays identified in
the figure, and useful for PIXE analyses

155
The probability of producing the X-ray photons of a particular line, or X-ray production cross section,
is a physical magnitude that depends on several factors, such as the projectile, its energy, and the
target atom. For the K lines, it is related to the probability of ionizing the atom, or ionization cross
section, by the equation

σ X ,i = σ I ,K ωK Pi , (1)

where σX,i is the X-ray production cross section, σI,K is the K-shell ionization cross section, ωK is the
fluorescence yield, and Pi is the relative intensity of all the possible transitions included in the line i.
The fluorescence yield is the ratio of the number of emitted X-ray photons to the number of total
produced vacancies in the K shell. For the L shell (and upper shells), the expressions are more
cumbersome because, besides the quantity of radiative transitions of electrons coming from higher
shells, it is possible to have non-radiative transitions form different subshells within the shells L1, L2,
and L3. If a vacancy is created in the L1 subshell, it may be filled by an electron from the L2 subshell,
which in turn may be filled by an electron coming from the L3 subshell. The net result is a vacancy in
L3. These processes are known as Coster-Kronig transitions. Furthermore, it is necessary to know the
ionization cross sections and fluorescence yields for each subshell. As a result, it is possible to obtain
for the L X-ray production cross sections of the most common lines the following equations:

σ X ,Lα = (σ L f13 + σ L f12f23 + σ L f23 + σ L )ω3 F3α ;

1 1 2 3
(2)

σ X ,Lβ = σ L ω1F1β + (σ L f12 + σ L )ω2F2β + (σ L f13 + σ L f12f23 + σ L f23 + σ L )ω3F3 β ; (3)

1 1 2 1 1 2 3

σ X ,Lγ = σ L ω1F1γ + (σ L f12 + σ L )ω2F2γ ;

1 1 2
(4)

σ X ,L = (σ L f13 + σ L f12f23 + σ L f23 + σ L )ω3F3l ,

l 1 1 2 3
(5)

Where the σLi are the ionization cross sections of an individual Li subshell, ωi is the fluorescence yield
of the i subshell, fij is the probability for a Coster-Kronig transition between subshells i and j, while Fnx
is the probability of producing a radiative transition, taken as the fraction of of X-ray photons
originated from a vacancy in the Ln subshell, contributing to the Lx line.
As can be seen from eqs. (2) to (5), it is necessary to know the ionization cross sections for each
individual subshell. However, sometimes it is also possible to define an average fluorescence yield ωL,
and to use a relative intensity of the line i, in analogy to the case of the K shell. Therefore,
σ X ,L = σ I,LωL Pi ,
i
(6)

equation where σI,L is the total ionization cross section for the L shell, ωL is the average fluorescence
yield, and Pi is the relative intensity of line Li. The use of this equation is limited, because the relative
intensity depends on the projectile energy.

2.- The Ionization Cross Section

From the preceding section it is apparent that one of the most important physical magnitudes for the
application of PIXE is the ionization cross section. Nevertheless, this is not a magnitude easy to
compute, although several models for its prediction have been developed along the years. Among the
most important theories, it is possible to mention:
• The Binary Encounter Approximation (BEA);

156
 • The Semiclassical Approximation (SCA);
• The Plane Wave Born Approximation (PWBA);
• The ECPSSR correction to the PWBA model.
There will be a description of these models in the following.

2.1.-The Binary Encounter Approximation (BEA)

It is possible to describe classically the phenomenon of inner shell ionization by the BEA. In this
theory, the collision of the projectile and a complex atom can be sketched as the binary encounter of
the projectile and a particular electron in an atomic energy level. The rest of the atom is considered
only as a passive environment in which the electron is moving.
THE BEA is based on the fact that the differential cross sections in the center-of-mass frame for
Coulomb scattering are identical both in the classical and quantum descriptions. The prediction of the
ionization in this theory is completely inappropriate in the low projectile energy regime, and thus its
use is not very extended.

2.2. -The Semiclassical Approximation (SCA)

This model is computed using a reference frame located on the target atom; the Coulomb interaction
between projectile and target is considered as time-independent. The basis of this theory is the
assumption of a rectilinear trajectory of the incoming ion (a purely classic concept), and from here to
calculate the probability for electronic transitions between different states. For ionization, the final
state would be the continuum.
As expected, this model is not valid for all the energy and projectile-target combinations, because of
the straight trajectory approximation. There are several improvements, however, that consider
hyperbolic trajectories, providing better results. Furthermore, this is the only model that gives a
dependence of the ionization cross section with the projectile impact parameter.

2.3.- The Plane Wave Born Approximation (PWBA)

In this model (Madison and Merzbacher, 1975), both the incoming and scattered ion are taken as plane
waves, according to a quantum approach. In this regard, the target atom appears as a scattering center,
in such a way that the Born scattering model can be applied. For this approximation to be valid, there
are three requirements:
i. The projectile acts as a point charge;
ii. The initial and final waves of the projectile are plane in all the space;
iii. The electron states (initial and final), are those of the unperturbed atom.
To fulfill these conditions, the projectile charge must be small, and its velocity rather large. Because of
this, the use of the PWBA is strongly restricted. Moreover, for the evaluation of total ionization cross
sections, the SCA and the PWBA are equivalent.

2.4.- The ECPSSR Theory

The ECPSSR theory is a substantial improvement to the PWBA and SCA (Brandt and Lapicki, 1981).
This model takes into account the ion energy loss during the collision (E), the Coulomb deflection of
the projectile (C), the increase in the binding energy of the ejected electron, described through the
Perturbed Stationary States method (PSS), and the relativistic mass of the involved electron (R).

157
This is the model that fits best all the experimental results; because of this, it is the most widely used
scheme for analytical applications using PIXE.
It is possible to observe the values predicted by the ECPSSR theory for the ionization of Cu by proton
impact, as a function of the projectile incident energy, in Figure 3. The curve corresponds to semi-
empirical calculations, based on the comparison of experimental data with the ECPSSR predictions.
Ionization Cross Section (b)

Proton Energy (MeV)

Figure 3. Ionization cross section of Cu as a function of proton incident energy

3.- Quantitative Analysis Using PIXE

To perform quantitative analyses using PIXE, typically three kinds of targets are considered: thin,
thick, and with intermediate thickness [Johansson and Campbell, 1988]. The difference among the
three types lies on the stopping of the ions inside the target and the attenuation in the sample of the
emitted X-rays. For a thin target it is possible to neglect both phenomena; in a thick target, the
projectile is stopped completely and the X-ray attenuation is important. In the case of an intermediate
target, the ion energy loss in the sample is high, although not total, while the X-ray attenuation is non-
negligible. In the following sections, a description of these approaches is given.

3.1.- Thin Target

As explained above, the simplest description for PÏXE quantitative analysis is that of a thin target. In
this case, the projectile loses very little energy (not more than 2%-3%), when crossing the sample,
while the X-ray attenuation in the target itself can be completely neglected. Therefore, in this
approximation, the number of X-ray photons Y0 for a certain X-ray line of an element with atomic
number Z is:

α
N P M (Z ) σ Z (E 0 )ω Z bZ ε Z N Av
Y0 ( Z ) = , (7)
S AZ

158
where NP is the number of ions that impinged on the sample, M(Z) is the mass of element Z, AZ is the
atomic mass of the element, ωZ is the fluorescence yield, bZ is the fraction of K or L X ray photons
emitted as Kα or Lα, εZ is the absolute efficiency of the detection system for the line of interest, NAv is
Avogadro’s number, σZ(E0) represents the ionization cross section at the incident ion energy E0, while
S is the beam cross section, assumed as being uniform.
Very often it is useful to define the thin target sensitivity, k(Z), as follows:

σ Z (E 0 )ω Z bZα ε Z N Av
k (Z ) = , (8)
AZ

in such a way that eq. (7) becomes

Y0 (Z ) = k ( Z )N P M a (Z ) , (9)

where

M (Z)
M a (Z ) = (10)
S

is the surface mass density of the element analyzed with PIXE.

3.2.- Intermediate Thickness Target

The second approximation appears when the ion energy loss and X-ray attenuation in the target cannot
be neglected, as in a thin target, even if the ion beam can pass through the sample. This is the case of
intermediate thickness targets. To establish the equations needed for this analysis, it is necessary to
look at Figure 4. There, an interaction generating X-rays in a target of thickness t, small but finite, is
shown. Along the path that goes from x to x + dx, the energy decreases from E to E – dE, with a
relationship given by the equation

E
dE
x= ∫ ρ S (E ) ,
E0
(11)

where S(E) is the ion stopping power (Ziegler et al., 1998). If ρ is the density of the target matrix, ρZ
as the density of the trace element to be analyzed, the ρZ/ρ is the concentration CZ of the element.
Therefore, the mass surface density of the element in the slice (x, x + dx) is

C Z dE
M a (Z ) = ρ Z dx = , (12)
S (E )

159
And using eq. (7), the number of X-ray photons is

N P C Z σ Z (E )ω Z bZα ε Z N Av e − µ x / cos θ dE
dY (Z ) = . (13)
S(E )AZ

Alter integration of this equation and using the expression for a thin target, a correction factor for the
intermediate thickness target is found:

Y0 (Z ) ρ t σ Z (E 0 )
F (Z ) = = . (14)
Y (Z ) E
σ Z (E )e − µ x / cos θ
∫
E0
S (E )
dE

Ion

X-ray
Sample
Photon
Figure 4. Geometry for quantitative analysis of an intermediate thickness target

This correction factor can be evaluated only if the matrix composition is known or assumed with
accuracy, as the stopping power and the X-ray attenuation must be calculated.
In short, to carry out intermediate thickness target analysis, it is required to obtain the thin target
sensitivity according to eq. (8), to obtain elemental concentrations as if the target is thin, and to
multiply by the factor given in eq. (14), obtaining through this procedure the intermediate thickness
target concentrations.

3.3.- Thick Target Analysis

The third case of interest for quantitative PIXE analysis corresponds to thick targets, namely, when the
projectile deposits all its energy inside the sample, so it is fully stopped. Thus, it is necessary to have a
complete knowledge of the ion stopping inside the material, as well as the X-ray attenuation.
Taking Figure 5 as a reference, it is possible to obtain the equation for the total number of X-ray
photons produced by the ion along its trajectory inside the material, in a very similar fashion as that of
the intermediate thickness target, except that in the present case the lower integration limit for the
energy is zero or a very small value, when the X-ray yield is negligible. Therefore, for the number of
X-ray photons produced by element Z, it is found that

160
 N Av ω Z bZα ε Z N P C Z 0 σ Z (E )TZ (E )
Y (Z ) =
AZ ∫ S(E ) dE ,
E0
(15)

where all the symbols were already defined. Except for the term TZ(E), which describes the
transmission of the photons inside the target. It is given by

Sample
Interaction spot

X-ray
α
Photon

π/2−θ
To
Ion Detector

Figure 5. Geometry for quantitative analysis of thick targets

⎧⎪ ⎛ µ ⎞ cos α E
dE ⎫⎪
T Z (E ) = exp⎨− ⎜⎜ ⎟⎟
⎪⎩ ⎝ ρ ⎠ Z senθ T0
∫ S(E ) ⎬⎪ . (16)
E0 ⎭
The mass attenuation coefficient (µ/ρ)Z for the X-rays of element Z in the sample matrix is obtained as
follows:

⎛µ⎞ NE
⎛µ⎞
⎜⎜ ⎟⎟ = ∑ C i ⎜⎜ ⎟⎟
⎝ ρ ⎠ Z i =1 ⎝ ρ ⎠ Z ,i , (17)

Where Ci is the concentration of element i, while (µ/ρ)Z,i is the mass attenuation coefficient of X-rays
from element Z in the pure element i.
The quantitative analysis of thick targets must be carried out by, first, measuring the number of X-ray
photons, and then computing numerically eqs. (15) and (16), iterating until the numerical results agree
with the experimental measurements.
An important effect occurring in thick target analyses (and sometimes in intermediate thickness
samples) is the appearance of secondary fluorescence. This phenomenon is due to the emission of X-
rays from an element that may produce ionization in atoms of a second element (via the photoelectric
effect). The energy of the X-rays from the first element is slightly higher than the binding energy of
the inner-shell electrons from the second element. Thus, a vacancy in the latter element is created,
with the possibility of emitting X-rays from this element. For a complete and accurate analysis, then it
is necessary to include this secondary fluorescence effect in the evaluation of eqs. (15) and (16), as the
number of X-ray photons is increased. An example of this situation is the analysis of stainless steel,
where the X-rays from Fe enhance the production of X-rays from Cr.

161
4.- Experimental Setup for PIXE Analysis
The experimental setup used in PIXE analyses depends upon the kind of sample to be studied.
Therefore, in the case of thin target analysis, a system as that sketched in Figure 6 may be used. There,
the beam current is measured in a Faraday cup located behind the target, as the ion beam can traverse
it. The difference with the thick target analysis device (Figure 7) is that the current must be measured
on the sample itself, if it is a conductor, or on the irradiation chamber as a whole, when the sample is
an insulator. Other indirect methods, such as the detection of backscattered ions with a particle
detector, are often used.
The main constituent of a laboratory for PIXE analysis is a particle accelerator, typically one of the
several electrostatic kinds (Scharf, 1989). The accelerator produces the ion beam, most commonly a
proton beam with energies between 1 MeV and 3 MeV, which induces the X-rays from the irradiated
sample. The energy and mass of the beam is analyzed with a switching magnet. The emitted X-rays
are usually registered with a semiconductor detector (lithium-drifted silicon or high purity
germanium), so PIXE is an energy-dispersive spectroscopy. The signal produced by the detector is
processed by a preamplifier and an amplifier, creating a spectrum collected with a multichannel
analyzer.
When the sample is an insulator, the beam charge is frequently accumulated in the sample, producing
a high voltage, which can produce background radiation (Section 5), damaging the signal-to-noise
ratio in the spectra, and thus impoverishing the detection limits. The solution to this problem is the use
of a small electron gun (which may be only a carbon filament with a grid at a 100 V potential), to
irradiate simultaneously the sample, neutralizing the positive charge from the ion beam.
Furthermore, it is possible to analyze samples that are unusually large or that cannot be introduced in a
vacuum chamber (for example, art works, archaeological objects, organic materials or liquid samples).
In this case, it is possible to use an external beam setup. Here, a thin window is placed at the end of
the beam line; the material must be radiation resistant, like Kapton® or aluminum (with a thickness
around 5 µm). This way, the beam can traverse this thin foil and then impinge on the sample to carry
out the PIXE analysis. There is a diagram of such a device in Figure 8.

X-ray
Detector
Ion
Sample
Accelerator Particle
Switching
Detector
Magnet

Faraday
Faraday Cup and Cup A
Nuclear Electronics Beam Profile
Monitor
Current
Multichannel Integrator
Analyzer

Figure 6. Experimental setup for PIXE analysis of thin targets

162
A further improvement to the analytical capabilities of PIXE is the use of an ion microprobe,
microbeam or nuclear microscope (Breese et al., 1996; Llabador and Moretto, 1998). Here, a set of
magnetic lenses is used to focus the ion beam image of an aperture (called the object aperture) onto the
sample under study. So, it is possible to have ion beams with diameters around 1 µm. Then, a device
used to scan the beam on the sample’s surface is included, to obtain a bi-dimensional mapping of the
elemental concentrations.

X-ray
Detector
Ion
Sample
Accelerator Particle
Switching
Detector
Magnet

Faraday Cup and A

Nuclear Electronics Beam Profile
Monitor
Current
Multichannel Integrator
Analyzer

Figure 7. Experimental setup for PIXE analyses of thick targets

Lasers for
Ion Accelerator Switching Sample
Magnet Alignment
8 µm Al
Window
Proton Beam

Sample
Multichannel
X-ray
Analyzer X-rays
Detector

Amplifier
Figure 8. Schematics of an external beam device for PIXE analysis

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5.- Background Radiation
Every PIXE spectrum contains, besides the characteristic X-ray peaks, a background radiation. The
origin of this radiation may be multiple: braking radiation (bremsstrahlung) from the ion beam or
secondary electrons produced by the sample (Ishii and Morita, 1990); radiation scattering in the
detector (Compton scattering) of the characteristic X-ray photons produced by the target atoms; or
gamma radiation produced by other nuclear interactions between the ions and the target atoms (as is
the case of protons interacting with fluorine nuclei). All these factors affect the quality of a PIXE
spectrum, which must then be analyzed with computer programs. This way, the main components of
the spectrum are the characteristic peaks and the background continuum.

5
10
K Cr Fe
Sediment Lake SL-1
4
Ca Mn
10 Ar Ti Ni
X-ray Yield (Arbitrary Units)

3
Zn
10
Cu
Ga Rb
2
10 As Pb
Sr Zr

1
10

0
10
0 5 10 15 20
Energy (keV)

Figure 9. PIXE spectrum for a sample of International Atomic Energy Agency Standard Reference
Material Sediment Lake SL-1, obtained with an external beam setup using a 2.7 MeV proton beam. The
element emitting each X-ray peak is marked

6.- Applications
Several examples of applications of PIXE in various scientific areas (environmental sciences, food
chemistry, and Archaeometry) are presented in this section, to illustrate the capabilities of PIXE.

6.1.- Environmental Sciences

In this field, several studies regarding elemental analysis of atmospheric aerosols from several urban
areas (Miranda, 1996; Miranda et al., 2004a), or the study of contaminated soils (Huerta-Arcos et al.,
2002).
The importance of atmospheric aerosols comes from the various effects caused by them (Cahill, 1996).
In the first place, although many efforts have been made to determine if the carbon dioxide has
produced an increase in the global temperature (greenhouse effect), atmospheric aerosols are a factor
not fully considered. The particles suspended in the atmosphere may have an effect opposite to that
produced by carbon dioxide, reducing the global temperature due to scattering and reflection of light

164
from the Sun. Thus, as long as the effects of aerosols are not deeply understood, it will be very
difficult to define if there is, indeed, a global warming.
Furthermore, many of the particles have dimensions below 10 µm, and can penetrate the human
respiratory tract. The smallest particles may even reach the pulmonary alveoli, with important health
effects. Also, aerosols with sizes below 2.5 µm are very efficient to scatter visible light. This has as a
result a decrease in visibility. This not limited, though, to urban areas; the reduction of visibility has
been observed in the USA national parks (Johansson and Campbell, 1988). Finally, there is a strong
effect of aerosols in buildings and monuments. As the aerosols include components that may react
with the materials of the building, there is a slow degradation. An example of this is the Parthenon in
Athens, Greece, which suffered a more severe damage in the past 25 years than in the previous
centuries, as the aerosol concentrations are higher in the present. All this only explains, in a shallow
manner, the interest created by atmospheric aerosols, both in urban and remote areas.
These facts justify the development of several studies of aerosols in the Mexico City Metropolitan
Area (MCMA) (Miranda et al., 1994; Miranda et al., 2000; Miranda et al., 1998; Miranda et al.,
2004b). An example of the information obtained in these studies is given on Figure 10. There, a time
series for the behavior of elemental contents in atmospheric aerosols with dimensions below 2.5 µm,
in the MCMA, along the first half of the year 2002, is displayed. Fourteen elements were found, from
S to Pb. Also, several episodes occur along this period, associated to emissions from industry or the
neighboring Texcoco Lake, which is a major source.
Another of the advantages of PIXE is the possible application of receptor models, such as Absolute
Principal Component Analysis (APCA), which has been utilized successfully in the MCMA (Miranda
et al., 1994; Miranda et al., 2000). This statistical method provides information regarding emitting
sources of the different elements.
However, it is necessary to emphasize that PIXE does not offer a complete analysis of the composition
of atmospheric aerosols. Thus, other complementary techniques must be used, as the composition is so
complex, that only about 20 % of the total aerosol mass is studied with PIXE; the remaining mass
corresponds to organic compounds or particles.
The studies performed with PIXE along 15 years allowed to observe a decrease in the concentration of
certain elements (Zn and Pb), although other elements remained constant along this period (as is the
case of S).

4000

S
3500 Cl
K
Ca
3000 Ti
Concentration (ng m )

V
-3

Cr
2500 Mn
Fe
Ni
2000 Cu
Zn
Se
Pb
1500

1000

500

0
10 May
16 May
22 May
28 May
4 May
16 Jan
22 Jan
28 Jan
3 Feb
9 Feb
15 Feb
21 Feb
27 Feb

3 Jun
9 Jun
15 Jun
21 Jun
27 Jun

15 Jul
21 Jul
27 Jul
3 Jul
9 Jul
5 Mar
11 Mar
17 Mar
23 Mar
29 Mar
4 Apr
10 Apr
16 Apr
22 Apr
28 Apr

Figure 10. Time series of elemental composition of atmospheric aerosols in the North of Mexico City, in
the year 2002

165
6.2.- Food Chemistry
An extensive knowledge about the composition of food is necessary, due to possible effects that some
elements they may have in human health. Although some elements are considered as essential for life,
(such as P, S, K, Ca, Ti, Cr, Mn, Fe, Cu, and Zn), their excess may be harmful, as they may surpass
the tolerance limits of the human body. Therefore, measurements of the trace element contents in food
for human consumption are required. There are several reasons why some elements are present in
food. For instance, it may be a result of natural biological process or pollution in the environment
where they were grown. Processed food may be contaminated during the packing and handling, or
during the preparation procedure. Furthermore, some elements may be added intentionally, as
additives.
With this in mind, it was considered necessary to study the elemental contents in several kinds of food,
either fresh or processed, using PIXE. Examples of these studies were those carried out on tomato
puree, edible cactus, and chocolate. When possible, comparisons were made among brands form
Mexico and other countries.
To show only an example, the results obtained for tomato puree (Lycopersicum esculentum P. Mill),
are explained (Romero-Dávila and Miranda, 2004). In this case, samples of 12 brands available in the
Mexican market were collected, together with three brands form the U.S.A., two from Chile, one from
Colombia, and one more from Japan. Also, a reference sample was prepared, according to the method
suggested by the Mexican Federal Consumer Attorney (Profeco). As a whole, 20 samples were
studied.
The specimens were dried, using first ethanol, and then heating a temperature of about 50 °C; the
resulting material was ground with an agate mortar and pressed to obtain a pellet.
All the samples were analyzed with PIXE, using the external beam setup at the Pelletron accelerator,
Instituto de Física, UNAM. A 3 MeV proton beam and both a Si-PIN diode and a high purity Ge
detectors were employed.
The results of PIXE analyses showed the presence of 13 elements: P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu,
Zn, Rb, and Sr, with strongly variable concentrations. Table 1 summarizes the concentration ranges
found for each element.

Element Concentrations (wet basis)

P 0.01-0.33 %
S 0-0.096 %
Cl 20-2300 mg/kg
K 20-1900 mg/kg
Ca 0.2-1100 mg/kg
Ti 0-3 mg/kg
Cr 0-0.5 mg/kg
Mn 0-1 mg/kg
Fe 0-2 mg/kg
Cu 0-0.7 mg/kg
Zn 0-0.7 mg/kg
Table 1. Ranges of elemental concentrations measured in different brands of tomato puree (Romero-
Dávila and Miranda, 2004)

166
The conclusions of this study were that none of the elemental concentrations of the several tomato
puree brands exceeds the standards, either national or international.

6.3.- Archaeometry
In the case of Archaeometry, that is, the application of sciences like Chemistry and Physics in
archaeology, many works have been carried out. Among them, it is possible to cite studies about
prehistoric wall paintings and obsidians from Baja California Sur, Mexico (Miranda et al., 1993);
stuccos from the site of Xochicalco, in central Mexico (Rodríguez-Lugo et al., 1999); the effects of
pollution on paintings of the Major Temple, Mexico City (Miranda et al., 1999); or the
characterization of ceramics from Teotihuacan (Ruvalcaba-Sil et al., 1999; Ontalba Salamanca et al.,
2000).
The main obstacle with archaeological samples is that they are, usually, thick or intermediate.
Therefore, the beam energy may be deposited entirely on the target, with a high probability of causing
damage, so the ion beam intensity must be carefully regulated during the study. In this case, an
external beam setup is required. Additionally, when quantitative analysis is carried out, at least a rough
knowledge of the matrix composition must be given. For this, analyses with other complementary
techniques, like ion backscattering (Chu et al., 1978) or nuclear reaction analysis (Bird and Williams,
1989), can be used simultaneously with PIXE. This way, the integrals in eq. (15) can be computed
numerically.
In other cases, it may be useful to apply ion backscattering to determine the concentration of elements
that cannot be detected easily with PIXE. An example is given for Na and Mg, in fragments of
Prehispanic wall paintings from Baja California Sur (Miranda et al., 1993). Table 2 displays elemental
concentrations (in atomic percent) measured in samples of black and red paintings, as well as the
substrate. It is apparent that in the pigments (red and black) there is no Na or Mg. It was possible to
quantify the oxygen contents, too.

Element Red Black Substrate

O 62.7 69.7 53
Na - - 3.2
Mg - - 10.6
Al 3.83 4.25 4.7
Si 12.9 11.6 23.5
P 0.14 0.15 -
S 9.06 6.58 -
Cl 0.14 - -
K 0.7 0.77 0.59
Ca 8.36 5.03 1.53
Fe 2.16 1.93 2.8

Table 2. Elemental composition in a fragment of Prehispanic wall paintings, obtained with PIXE and ion
backscattering (atomic percent)

REFERENCES

167
Bird, J.R., and Williams, J.S., 1989. Ion Beams for Materials Analysis. Academic Press, San Diego.
Brandt, W. and Lapicki, G., 1981. Energy-loss effect in inner-shell Coulomb ionization by heavy
charged particles. Physical Review A 23: 1717-1729.
Breese, M.B.H., Jamieson, D.N., and King, P.J.C., 1996. Materials Analysis using a Nuclear
Microprobe. John Wiley, New York.
Cahill, T.A., 1996. Climatic forcing by anthropogenic aerosols: the role for PIXE. Nuclear
Instruments and Methods in Physics Research B 109/110: 402-406.
Chu, W.K., Mayer, J.W., and Nicolet, M.A., 1978. Backscattering Spectrometry. Academic Press,
New York.
Huerta-Arcos, L., Contreras-Valadez, R., Palacios-Mayorga, S., Miranda, J., and Calva-Vásquez G.,
2002. Total elemental composition of contaminated soils with wastewater irrigation by combining IBA
techniques. Nuclear Instruments and Methods in Physics Research B 189: 158-162.
Ishii, K. and Morita, S., 1990. Continous Background in PIXE. International Journal of PIXE 1: 1-30.
Johansson, S.A.E. and Campbell, J.L., 1988. PIXE: A Novel Technique for Elemental Analysis. John
Wiley, Chichester.
Johansson, S.A.E., Campbell, J.L., and Malmqvist, K.G., 1995. Particle Induced X-ray Spectrometry
(PIXE). John Wiley, New York.
Llabador, Y. and Moretto, P., 1998. Applications of a Nuclear Microprobe in the Life Sciences. World
Scientific, Singapore.
Madison, D.H. and Merzbacher, E., 1975. Theory of Charged-Particle Excitation. In. Crasemann, B.,
Ed. Atomic Inner-Shell Processes, Vol. I. Academic Press, New York. Pp. 1-72.
Maenhaut, W., and Malmqvist, K., 2002. Particle Induced X-ray Emission Analysis. In Van Grieken,
R.E. and Markowicz, A.A., Eds. Handbook of X-ray Spectrometry, 2nd Ed. John Wiley, New York. Pp
719-809.
Markowicz, A.A., 2002. X-ray Physics. In Van Grieken, R.E. and Markowicz, A.A., Eds. Handbook of
X-ray Spectrometry, 2nd Ed. John Wiley, New York. Pp 1-94.
Miranda, J., Oliver, A., Dacal, A., Ruvalcaba, J.L., Cruz, F. and Ortiz, M.E., 1993. PIXE Analysis of
Cave Sediments, Prehispanic Paintings and Obsidian Cutting Tools from Baja California Sur Caves.
Nuclear Instruments and Methods in Physics Research B75: 454-457.
Miranda, J., Cahill, T.A., Morales, J.R., Aldape, F., Flores M., J., and Díaz, R.V., 1994 Determination
of Elemental Concentrations in Atmospheric Aerosols in Mexico City Using Proton Induced X-ray
Emission, Proton Elastic Scattering and Laser Absorption. Atmospheric Environment 28: 2299-2306.
Miranda, J., 1996. Studies of Atmospheric Aerosols in Large Urban Areas Using PIXE: an Overview.
Nuclear Instruments and Methods in Physics Research B109/110: 439-444.
Miranda, J., López-Suárez, A., Paredes-Gutiérrez, R., González, S.,. de Lucio, O.G, Andrade, E.,
Morales, J.R., and Avila-Sobarzo, M.J. 1998. A Study of Atmospheric Aerosols from Five Sites in
Mexico City Using PIXE. Nuclear Instruments and Methods in Physics Research B137: 970-974.
Miranda, J., Gallardo, M.L., Grimaldi, D.M., Román-Berrelleza, J.A., Ruvalcaba-Sil, J.L., Ontalba
Salamanca, M.A., Rodríguez-Fernández, L., and Morales, J.G., 1999. Pollution Effects on Benches of
the Eagle Warriors Precinct of the Major Temple, Mexico. Nuclear Instruments and Methods in
Physics Research B150: 611-615.
Miranda, J., Crespo, I., and Morales, M.Á., 2000. Absolute Principal Component Analysis of
Atmospheric Aerosols in Mexico City. Environmental Science & Pollution Research 7: 14-18.
Miranda, J., Zepeda, F., Galindo, I., 2004a The Possible Influence of Volcanic Emissions on
Atmospheric Aerosols in the City of Colima, Mexico. Environmental Pollution 127: 271-279.

168
Miranda, J., Barrera, V.A., Espinosa, A.A., Galindo, O.S., Núñez-Orosco, A., Montesinos, R.C., Leal-
Caballero, A., and Meinguer, J., 2004b. PIXE analysis of atmospheric aerosols from three sites in
Mexico City. Nuclear Instruments and Methods in Physics Research B 219-220: 157-160.
Ontalba Salamanca, M.Á., Ruvalcaba-Sil, J.L., Bucio, L., Manzanilla, L., and Miranda J., 2000. Ion
Beam Analysis of Pottery from Teotihuacan, Mexico. Nuclear Instruments and Methods in Physics
Research B 161: 762-768.
Rodríguez-Lugo, V., Ortiz-Velázquez, L., Miranda, J., Ortiz-Rojas, M. and Castaño, V.M., 1999.
Study of Prehispanic Wall Paintings from Xochicalco, Mexico, Using PIXE, XRD, SEM and FTIR.
Journal of Radionalytical and Nuclear Chemistry 240: 561-569.
Romero-Dávila, E. and Miranda, J., 2004. Trace element determination in tomato puree using PIXE
and Rutherford Backscattering. J. Radionalytical and Nuclear Chemistry 262: 355-362.
Ruvalcaba-Sil, J. L., Ontalba Salamanca, M.A., Manzanilla, L., Miranda, J., Cañetas Ortega, J. and
López, C., 1999. Characterization of pre-Hispanic pottery from Teotihuacan, Mexico, by a combined
PIXE-RBS and XRD analysis. Nuclear Instruments and Methods in Physics Research B150: 591-596.
Scharf, W., 1989. Particle Accelerators and Their Uses. Harwood Academic Publishers, Chur,
Switzerland.

Javier Miranda
Instituto de Física. Universidad Autónoma de México
Apartado Postal 20-364. 01000. México, D.F. México
miranda@fisica.unam.mx

169
 10
Polymeric Materials by TXRF

Susana Boeykens

1.- Polymer Systems

Over the years, the term gums has been used to denote a wide range of compounds including
polysaccharides, terpenes, proteins, and synthetic polymers. In the 1990s, the term more specifically
denotes a group of industrially useful polysaccharides or their derivatives that hydrate in hot or cold
water to form viscous solutions, dispersions, or gels (1). Gums are used in industry because their
aqueous solutions or dispersions possess suspending and stabilizing properties. In addition, gums may
produce gels or act as emulsifiers, adhesives, flocculants, binders, film formers, lubricants, or friction
reducers, depending on the shape and chemical nature of the particular gum (2). Considerable research
has been carried out to relate the structure and shape (conformation) of some gums to their solution
properties (3,4).
Biopolymers have being used increasingly in recent years by industry due to their controlled,
reproducible and economical biosynthesis, and their biodegradability.
Specifications of these polymers have to be controlled by European Community, Mercosur, etc.
especially toxic metals in food and pharmaceutical products.
In this study the applicability of TXRF for the analysis of traces in aqueous media with high-viscosity
properties is shown.
Polymers used in this work
Gums are classified as natural or modified. Natural gums include seaweed extracts, plant exudates,
gums from seed or root, and gums obtained by microbial fermentation. Modified (semisynthetic) gums
include cellulose and starch derivatives and certain synthetic gums such as low methoxyl pectin,
propylene glycol alginate, and carboxymethyl and hydroxypropyl guar gum. For this work we have
selected polymers that correspond to these different types.

170
Derivatives
Carboxymethylcellulose
Carboxymethylcellulose (CMC) is a derivative of cellulose formed by its reaction with alkali and
chloroacetic acid. The CMC structure is based on the β-(1→4)-D-glucopyranose polymer of cellulose
(Figure1). Different preparations may have different degrees of substitution, but it is generally in the
range 0.6 - 0.95 derivatives per monomer unit.

Figure 1. Monomer unit of CMC

CMC molecules are somewhat shorter, on average, than native cellulose with uneven derivatization
giving areas of high and low substitution. CMC molecules are most extended (rod-like) at low
concentrations but at higher concentrations the molecules overlap and coil up and then, at high
concentrations, entangle to become a thermoreversible gel. Increasing ionic strength and reducing pH
both decrease the viscosity as they cause the polymer to become more coiled (5).
CMC dissolves rapidly in cold water and mainly used for controlling viscosity without gelling (CMC,
at typical concentrations, does not gel even in the presence of calcium ions). As its viscosity drops
during heating, it may be used to improve the volume yield during baking by encouraging gas bubble
formation. Its control of viscosity allows use as thickener, phase and emulsion stabilizer (e.g. with
milk casein), and suspending agent. CMC can be also used for its water-holding capacity as this is
high even at low viscosity; particularly when used as the Ca2+ salt. Thus, it is used for retarding staling
and reducing fat uptake into fried foods.
The average chain length and degree of substitution are of great importance; the more-hydrophobic
lower substituted CMCs are thixotropic but more-extended higher substituted CMCs are
pseudoplastic. At low pH, CMC may form cross-links through lactonization between carboxylic acid
and free hydroxyl groups.
The colorless, odorless, nontoxic, water-soluble powder or granules are stable in pH range 2 – 10, and
Insoluble in organic liquids. They react with heavy-methal salts to form films that are insoluble in
water, transparent and unaffected by organic materials.
CMC is used in detergents, soaps, food products (especially dietetic foods and ice cream), where it
acts as water binder, thickener, suspending agent, and emulsion stabilizer, textile manufacturing
(sizing); coating paper and paper board to lower porosity, drilling muds, emulsion paints, protective
colloid, pharmaceuticals, cosmetics.
Algal (Seaweed) Gums
Sodium alginate
Algin, the generic description for salts of alginic acid, was discovered about 1880 in the United
Kingdom (6). Commercial production started in 1929 in California. The development of propylene
glycol alginate in 1944 increased the utility of algin in both food and general industrial applications.
Algin occurs in all members of the class Phaeophyceae, brown seaweed, as a structural component of
the cell walls in the form of the insoluble mixed calcium, magnesium, sodium, and potassium salt of
alginic acid.

171
Structure:
Alginate is a linear copolymer composed of two monomeric units, D-mannuronic acid and L-guluronic
acid (Figure2).
These monomers occur in the alginate molecule as regions made up exclusively of one unit or the
other, referred to as M-blocks or G-blocks or as regions in which the monomers approximate an
alternating sequence. The calcium reactivity and gelling properties of alginates are a consequence of
the particular molecular geometries of each of these regions (7). Because of the particular shapes of
the monomers and their modes of linkage in the polymer, the geometries of the G-block regions, M-
block regions, and alternating regions are substantially different. Specifically, the G-blocks are
buckled whereas the M-blocks have a shape referred to as an extended ribbon. If two G-block regions
are aligned side by side, a diamond-shaped hole results. This hole has dimensions that are ideal for the
cooperative binding of calcium ions. When calcium ions are added to a sodium alginate solution, such
an alignment of the G-blocks occurs and the calcium ions are bound between the two chains like eggs
in an egg box. Thus the reactivity of algins is the result of calcium-induced dimeric association of the
G-block regions. The detailed chemical structure has been determined by NMR spectroscopy.

Figure 2. Monomer unit of sodium alginate

Alginates available for industrial use are in general alginate metal salts. These water-soluble alginates
are produced in many forms varying in molecular weight, calcium content, particle form (granular or
fibrous) and particle size distribution.
The ability of alginates to form edible gels by reaction with calcium salts is an important property.
Commercially available alginates dissolve in hot or cold water to produce solutions with viscosities
ranging from a few to several hundred mPa.s. The actual viscosity depends on the molecular weight
and calcium content of the alginate.
The viscosity of alginate solutions decreases with increasing temperature, but provided the
temperature is not maintained at high levels for extended periods, the viscosity decrease is reversible.
Partial depolymerization of the alginate occurs if solutions are exposed to excessive temperatures or to
sufficiently elevated temperatures for extended periods.
The viscosity of alginate solutions is independent of pH in the range 5-10, but below pH 4.5, the
viscosity increases until the pH reaches 3 when insoluble alginic acid precipitates. The viscosity of
sodium alginate solutions is slightly depressed by the addition of monovalent salts. As is frequently
the case with polyelectrolytes, the polymer in solution contracts as the ionic strength of the solution is
increased. The maximum viscosity effect is obtained at about 0.1 N salt concentration.
The flow properties of sodium alginate solutions depend on concentration. A 2.5% medium viscosity
sodium alginate solution is pseudoplastic, especially at the higher shear rates in the range of 10-
10000/s.
All species of brown algae contain algin; however, most of the algin produced commercially is
isolated only from a few species.

172
Alginates are used in a wide range of applications, particularly in the food, industrial, and
pharmaceutical fields. These applications arise from the properties of gelation, thickening/water
holding, emulsification, stabilization/binding, and film forming.
Carrageen
The term carrageenan is the generic description for a complex mixture of sulfated polysaccharides that
are extracted from certain genera and species of the class Rhodophyceae, red seaweed.
Residents of County Carragheen on the south coast of Ireland are reported to have used a seaweed in
foods and medicines about 600 years ago. This seaweed became known as Irish moss. The extraction
and purification of the polysaccharide from Irish moss was patented in 1871(8). This polysaccharide
eventually became known as carrageenan; it was not produced and marketed until 1937.
Commercial carrageenan has a molecular weight in the range of 100,000 to 1,000,000. It is sold as
powder ranging from white to beige, depending on the grade. Carrageenan produces high viscosity
solutions and gels in water which react with proteins, especially casein. This allows the preparation of
high strength milk gels. Most of the uses for carrageenan are in the food industry. A small amount of
carrageenan is used in cosmetics and pharmaceuticals.
Carrageenan is a mixture of galactans that carry varying proportions of half-ester sulfate groups linked
to one or more of the hydroxyl groups of the galactose units (9). The repeating galactose units are
joined by alternating β-1,3 and β-1,4 glycosidic linkages. In addition, the β-1,4-linked galactose units
often occur as 3,6-anhydro-D-galactose (Figure 3).

Figure 3. Monomer unit of carrageen

The sulfate groups are believed to be important in gelation with potassium and in reaction with
protein. The 3,6-anhydrogalactose units increase hydrophobicity and reduce solubility whereas the
sulfate groups increase hydrophilicity and solubility.
The shape or conformation of carrageenan has been determined (6) and related to the gelation
mechanism.
Several genera and species of red algae contain carrageenan, including Chondrus crispus, Eucheuma,
and Gigartina stellata. Red algae are abundant on the northeast coast of the North American continent.
Eucheuma species grow in tropical waters extending from the Philippines to the east coast of Africa.
The seaweed is harvested by raking and hand-gathering. Mechanized harvesting or use of divers has
met with limited success. The collected seaweed is dried mechanically in many areas and shipped to
the processing plants.
Processing conditions are closely guarded trade secrets, but manufacturer's literature provides some
information. The seaweed is extracted with hot water at slightly alkaline pH. The aqueous extract is
filtered and recovered by alcohol precipitation, dried, and milled. Drum-drying provides a less pure
product.
The use of carrageenan in foods is dependent upon the properties of solubility, viscosity, gelation,
reactivity with proteins, and synergism with the nongelling polysaccharide, locust-bean gum. Because
of its reactivity with certain proteins, carrageenan has found use at low concentrations (0.01-0.03%) in

173
a number of milk-based products, such as chocolate milk, ice cream, puddings, and cheese analogues.
It is also used in bakery gels and icings, low sugar jams and jellies. Because of its reactivity with
proteins, it is used in ham pumping, an operation in which commercial hams are injected with a
carrageenan and phosphate solution to retain moisture on cooking.
Botanical Gums
The botanical gums represent a family of polysaccharides obtained from a wide variety of plant
supplies. They are subdivided into exudate gums, seed gums, and gums obtained by extraction of plant
tissue. For a gum to be used in commercial quantities, it must be present in the tissues or be readily
extractable in relatively pure form that limits the number of commercial botanical gums. The
considerable viscosity variation observed among gums from different Supplies determines, in part,
their uses.
Plant Exudates
Most plant families include species that exude gums, and those that produce copious quantities
represent a ready supply of gums. These exudates were the first gums to be used commercially and
still represent a significant, but diminishing segment of the natural gum market. The plants are usually
shrubs or low growing trees. Collection is by hand and labor costs represent a large proportion of the
cost of these gums.
The quality of individual gums is mainly determined by color and taste or odor. Many gums are
colorless when secreted, but darken on aging. Most gums are usually tasteless unless contaminated by
the bitter flavors of tannins which precludes their use in foods.
Pectine
Pectin is a generic term for a group of polysaccharides, mainly partially methoxylated
polygalacturonic acids (Figure 4), which are located in the cell walls of all plant tissues. The main
commercial Supplies of pectin are citrus peel and apple pomace, where it represents 20-40% and 10-
20% of the dry weight respectively. The pectin is extracted, the extract purified, and the pectin
precipitated (10); increased extraction times lead to the production of low methoxyl pectins.

Figure 4. Pectine monomer unit

Pectins are readily soluble in water to give viscous stable solutions. However, the importance of pectin
to industry, in particular the food industry, is the ability of its solutions to form gels with sugar (ca
65% solids). Pectins are generally classed according to their ester content as high methoxyl pectins
(>50% of the carboxyl groups esterified) or low methoxyl pectins (<50% of carboxyl groups
esterified).
Pectin preparations, particularly the high methoxyl ones, are used mainly for jams and jellies. The low
methoxyl pectins are of increasing utility in the preparation of low calorie, low sugar jams and jellies
as low methoxyl pectin-calcium gels. Pectins are also used as stabilizers and thickeners in frozen

174
desserts, fruit juice drinks, milk beverages, and in the preparation of restructured fruits and vegetables.
Industrial applications, in particular for paper and textiles, have been limited because of high cost.
Seed gums
Although most seeds contain starch as the principal food reserve, many contain other polysaccharides
and some have industrial utility. The first seed gums used commercially were quince, psyllium, flax,
and locust bean gum. However, only locust bean gum is still used, particularly in food applications;
quince and psylhum gums are only used in specialized applications.
Harvesting of these gums is expensive; however, harvesting from annual plants costs less than from
perennial plants or trees. This is clearly demonstrated by the tremendous increase in the use of guar, a
gum that is extracted from an annual leguminous plant. Since the beginning of commercial production
in 1953, the use of guar has risen rapidly. More guar gum is consumed than all other gums combined.
Guar Gum
Guar gum is derived from the seed of the guar plant, Cyamopsis tetragonolobus, a pod-bearing
nitrogen-fixing legume grown extensively in Pakistan, India. During processing, the seed coat is
removed by heating and milling. The endosperm, comprising approximately 40% of the seed, is then
separated from the germ by various milling processes. The final milled endosperm, which is
commercial guar gum, has a typical analysis of crude fiber, 2.5%; moisture, 10-15%; protein, 5-6%;
and ash, 0.5-0.8%.
Structurally, guar gum comprises a straight chain of D-mannose with a D-galactose side chain on
approximately every other mannose unit; the ratio of mannose to galactose is 2:1 (Figure 5)). Guar
gum has a molecular weight on the order of 220.000 Dalton (11).

Figure 5. Guar gum monomer unit

Guar gum hydrates in either cold or hot water to give high viscosity solutions. Although the viscosity
development depends on particle size, pH, and temperature, guar gum at 1% concentration fully
hydrates typically within 24 h at room temperature. Guar gum solutions are stable over the pH range
of 4.0-10.5 with fastest hydration occurring at pH 8.0. Solutions are somewhat cloudy because of the
small amount of insoluble fiber and cellulosic material present.
Guar gum is compatible with other common plant gums, starch, and water-soluble proteins such as
gelatin, except for the synergistic viscosity increases given by blends with xanthan gum. The presence
of salts, water-miscible solvents or low molecular weight sugars can affect the hydration rate.
As a result of its properties and low cost, guar gum is the most extensively used gum, both in food and
industrial applications. In the mining industry, guar gum is used as a flocculant or flotation agent,
foam stabilizer, filtration aid, and water-treating agent. In the textile industry, it is used as a sizing
agent and as a thickener for dyestuffs. The biggest consumer of guar gum is the paper industry where
it facilitates wet-end processing and improves the properties of the product.

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In the food industry, guar gum is applied as a stabilizer in ice cream and frozen desserts, and it is also
used as a stabilizer for salad dressings, sauces, frozen foods, and pet foods.
Gums obtained by extraction of plant tissue
Glucomannan
Glucomannan is a hydrocolloidal polysaccharide comprised of D-glucose and D-mannose residues
(hence, the name) bonded together in β-1,4 linkages (Figure 6).
Approximately 60% of the polysaccharide is made up of D-mannose. Some of the sugar residues in
glucomannan are acetylated. The molecular weight of this slightly branched polysaccharide ranges
from 200.000 Daltons to 2.000.000 Daltons.

Figure 6. Glucomannan monomer unit

Glucomannan, which is also classified as a soluble dietary fiber, is derived from konjac flour. Konjac
flour itself is derived from the Amorphophallus species, plants which are related to the common
philodendron house plant and which grow in only certain parts of the world, including some regions in
China and Japan. One member of the Amorphophallus genus, called Amorphophallus konjac, is also
known as voodoo lilly, devil's tongue and konjac. In addition to being known as konjac, the plant is
called ju ruo (pronounced in Chinese) by the Chinese people, and called konjaku or konnyaku by the
Japanese. It has a long history of use in both China and Japan as a food substance and as a folk
remedy. Glucomannan products are widely used in Japan and China as general health aids, topically,
for skin care and as a thickening agent for foods, among other things.
Glucomannan may have laxative activity. This effect is thought to be due to the swelling of
glucomannan with consequent increase in stool bulk. It may also have activity in the control of serum
glucose and lipid levels. It has putative bariatric activity.
Some studies indicate that glucomannan may improve glycemic control in Type 2 diabetics. The
mechanism of glucomannan's possible hypocholesterolemic activity is likewise, unclear. The
polysaccharide may stimulate the conversion of cholesterol to bile acids, as well as the fecal excretion
of bile acids. It may also decrease the intestinal absorption of cholesterol. The swelling of
glucomannan that occurs when it absorbs water in the gastrointestinal tract, may confer a feeling of
satiety in some and it has demonstrated some usefulness in the management of obesity, diabetes and
constipation. It has some favorable effects on lipids. Following ingestion of glucomannan, very little
of it is digested in the small intestine. Glucomannan is resistant to hydrolysis by the digestive
enzymes. Significant degradation occurs in the large intestine via the action of colonic bacteria. There
may be some absorption of these degradation products from the large intestine.
Microbial Gums
In the late 1950s and early 1960s, the possibility of producing fermentation gums, under controlled
conditions, was investigated. Many polysaccharides produced by microorganisms have been studied,
including xanthan gum, curdlan, bioalgin, pullulan, scleroglucan, and more recently, gellan, welan,
and rhamsan gums.
Xanthan
As a result of a project to transform agriculturally derived products into industrially useful products by
microbial action, the Northern Regional Research Laboratories of the USDA showed that the
bacterium Xanthomonas campestris produces a polysaccharide with industrially useful properties (12).

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Extensive research was carried out on this interesting polysaccharide in several industrial laboratories
during the early 1960s, culminating in commercial production in 1964.
As subsequently found for all microbial polysaccharides, the primary structure consists of regular
repeating units. Each unit contains five sugars: two glucose units, two mannose units, and one
glucuronic acid unit. The main chain of xanthan gum is built up of β-D-glucose units linked through
the 1- and 4-positions.

Figure 7. Xanthan monomer unit

The chemical structure of the main chain of xanthan gum is identical to the chemical structure of
cellulose. A three-sugar side chain is linked to the 3-position of every other glucose residue in the
main chain (Figure 7). About half of the terminal D-mannose residues contain a pyruvic acid residue
linked to the 4- and 6-positions. The distribution of these pyruvate groups is unknown. The
nonterminal D-mannose unit in the side chain contains an acetyl group at position 6.
Xanthan gum is a cream-colored powder that dissolves in either hot or cold water to produce solutions
with high viscosity at low concentration. These solutions exhibit pseudoplasticity, ie, the viscosity
decreases as the shear rate increases. This decrease is instantaneous and reversible. Solutions,
particularly in the presence of small amounts of electrolyte, have excellent thermal stability, and their
viscosity is essentially constant over the range 0 to 800°C. They are not affected by changes in pH
ranging from 2 to 10.
Xanthan gum dissolves in acids and bases, and under certain conditions, the viscosity remains stable
for several months. It has excellent stability and compatibility with high concentrations of many salts,
eg, 15% solutions of sodium chloride and 25% solutions of calcium chloride.
Xanthan gum is produced by the microorganism X campestris, originally isolated from the rutabaga
plant. The gum is produced commercially by culturing X campestris purely under aerobic conditions
in a medium containing commercial glucose, a suitable nitrogen source, dipotassium phosphate, and
appropriate essential elements. When the fermentation is complete, the gum is recovered from the
fermentation broth by precipitation with isopropyl alcohol, and dried, milled, tested, and packed.
Health and Safety Factors
The toxicological and safety properties of xanthan gum have been extensively investigated. On the
basis of these studies, the FDA issued a food additive order in 1969 that allowed the use of xanthan
gum in food products without specific quantity limitations.
The unique properties of xanthan gum make it suitable for many applications for the food,
pharmaceutical, and agricultural industries. Principal food applications include dressings, relishes,
sauces, syrups and toppings, puddings, dry mix products, cake mixes, beverages, dairy products, and
confectionery. It is also used in pharmaceutical suspensions and toothpaste. Industrial applications
include textile printing, pigment and dye suspensions, cleaners, and polishes. Oilfield applications
include drilling muds, workover and completion fluids, fracturing, and in enhanced oil recovery.

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Scleroglucan
Scleroglucan is secreted by the fungus Sclerotium Rolfsii. The chemical structure has been
characterized as a linear chain of linked β-(1,3)-D-glucopyranosyl units, with a single linked β-(1,6)-
D-glucopyranosyl unit to every third unit of the main chain (Figure 8). Scleroglucan dissolves in water
as a rod-like triple helix. Its average total length is about 1mm, its diameter is approximately 2nm and
it has a persistence length of 180nm. This neutral glucan has a mean molar mass 4.106Da (13). Its
solutions, even at low concentrations, attain a high viscosity and there is a tendency to form ordered
phases of the liquid crystal type (14).
Scleroglucan (Actigum CS11) is provided by Sanofi Bio Industries (France). To obtain the
homogeneous dispersions a measured mass of the solid is vigorously shaken during 24h in high-purity
water using a magnetic stirrer. The polymeric systems are purified following a protocol especially
tested and described in previous studies (15). The membranes used for dialysis were supplied by A.H.
Thomas Co., (No. 4465-A2).

Figure 8. Scleroglucan monomer unit

Synthetic polymers
With the objective to obtain comparative conclusions a synthetic linear polymer was used that
approximately contains half of oxygen atoms that the polysaccharides.
Polyoxyethylene (POE)
POE is a polyether consisting of a linear chain of oxyethyl monomer (Figure 9), is a non-ionic and
water soluble macromolecule. It is prepared by polymerization of ethylene oxide.

-[CH2-CH2-O]n-

Figure 9. POE monomer unit

In this polymer, the absence of intra or intermolecular hydrogen bridges reduces the possibility of
cluster formation so the solutions formed are much less complex from the microscopical point of
view.

2.- TXRF Advantages and Requirements

Instrumentation
We have previously proposed the applicability of Total Reflection X-ray Fluorescence analysis
(TXRF) for polymer systems (15,16). That technique is a variation of energy-dispersive X-ray
Fluorescence. A different optical arrangement makes the primary beam strike the sample at a glancing

178
angle of less than 0.1°. Because of this grazing incidence, the beam is totally reflected (17). TXRF is
used for chemical micro-, ultramicro- and trace analysis because of its high detection power and
sensitivity. Matrix effects cannot build up within the thin layer of the sample. By this technique, only
elements of atomic number higher than Si are detectable (18). All X-ray analysis was carried out at the
X-Lab in the National Atomic Comission, Argentina, using a total reflection system composed of an
X-ray spectrometer, an X-ray tube excitation system, a total reflection module and spectrum
acquisition and quantitation software. The X-ray spectrometer consisted of an 80mm2 Si(Li) detector
with 166eV of full width half-maximun for 5.9keV, a 0.008mm thick Be window, an Ortec 672 fast
spectroscopy amplifier and an analogical digital converter Nucleus PCA2. Excitation conditions were
40kV and 30mA in all cases.
The total reflection module designed at the Atominstitut der Östereichischen Universitäten, fitted with
a cut-off- filtered radiation from a fine focus diffraction molybdenum anode X-ray tube was employed
(19).
For spectral data analysis, the AXIL program was employed. The QXAS software package from the
International Atomic Energy Agency was employed for quantification data.

Sensitivity test

For these experiences, a set of calibration standard solutions was made, containing S, and I covering a
range between 5 and 50mgL-1. Co was chosen as internal standard owing to its non-interfering
properties. The system sensitivity required 0.5mgL-1 Co content. The fluorescent intensity of the Kα or
Lα lines was measured for each element. The acquisition time for each spectrum was 200s. For
measurements, an aliquot of 10ml of standard solution or sample was pipetted onto a quartz glass
sample carrier and allowed to dry to a thin film under an infrared lamp. Four replicates of each sample
were analysed. Intensity ratio between element i and internal standard (Co) was obtained by

where Ii is the line intensity (counts s-1) of the element i in the sample; Ci is its concentration (ng mL-
1
); ICo is the line intensity of the internal standard (Co) in the sample; CCo is its concentration; si is the
system sensitivity (counts mLng-1s-1) for the element i; sCo is the system sensitivity for the internal
standard (Co). Relative quantities (Ri (ng-1mL)) of the element i for this system are defined by

Sri; the no-dimensional relative sensitivity of the system for the element i, is defined by

that results in

179
Figure 10 shows the relative quantity (Ri) versus the analyte concentration (Ci) in the samples. The
slope of these curves, relative sensitivity (Sri) and the regression coefficient (r2) are also shown.

Figure 10: TXRF calibration curves for I and S. The relative intensities (Sr) and the correlation
coefficients (r2) are shown beside the curves

Detection limits

The detection limits (DL) were calculated (21) by measuring the element intensities in low
concentration solutions (as near to the background as possible).

where DLi is the minimum concentration (ng mL-1), that can be detected for the element i; and Bg is
the background intensity (counts s-1).

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Detection limits obtained by TXRF from aqueous and systems for K and L lines are presented in
Figure 11a and b, respectively. A small deterioration of the detection limits is observed between
aqueous and polymer systems. According to Figure 11, it can be considered that the increase in
concentration does not affect the detection limit.

Figure 11. Detection limits (pg) determined in aqueous (σ) and polymeric matrices (λ500 µg mL-1; υ5000
µg mL-1) for (a) K lines and (b) L lines, respectively

Internal standard vs Compton

Quantitative TXRF analyses are usually done by using the internal standard method. Due to the high
viscosity of these samples, a homogeneous distribution of the internal standard cannot easily be
achieved, and this is a precondition to obtain the reliable analytical results. To avoid inhomogeneities,
the use of the signal coming from the Compton scattering peak is proposed (16). This can be achieved,
because in viscous dispersions, the Compton peak signal is remarkably higher compared with those
coming from an aqueous solution, and as a consequence, statistically strong enough signal is available
as shown in Figure 12. Additionally, this analytical method has many advantages: short analysis times,
avoidance of the sample preparation, and lower cost. This procedure must be employed for polymeric
dispersions higher than 500µgmL-1 concentration.

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 Figure 12. Intensity ratio Compton/rayleigh coming from TXRF spectra of (a) aqueous matrix and (b)
polymer matrix.

Polymeric thin films

To satisfy the thin film condition for polymers, it was considered that the primary intensity is reduced
by only 10%, this means that the absorption is negligible, and taking into account that the primary
beam goes twice through the film, the thickness of the film, t, can be expressed as (21):
where: is the mass absorption coefficient of the entire matrix and ρ is the density of the film.

Assuming that:
(a) the wavelength of the incident radiation is 0.07nm (Mo Kα),
(b) the incident angle of the primary beam is 1.6mrad,
(c) the take-off angle of the fluorescent radiation is π/2,
(d) polymers are composed by C (40–50%), O (30–50%), and H (1–10%),
(e) the dry mass of the polymer is 0.2gcm-3 and the dispersion densities at 25°C are 1gcm-3.

The thickness t and the mass m of the polymer were experimentally obtained; the solvent is evaporated
assuming that an area of 12mm2 is covered by the sample. Results are shown in Table 1 considering
the following radiations emitted: KKα (Z = 19), 0.4nm wavelength; FeKα (Z = 28), 0.2nm wavelength;
SeKα (Z = 34), 0.11nm wavelength; and CdLα (Z = 48), 0.4nm wavelength. Results from Table 1
shows that the change in thickness and mass is a soft function of the fluorescent energy. This is due to
the fact that is mainly determined by the mass absorption coefficient of the incoming beam and
not for the emitted radiation. Usually, for working polymeric dispersions, the volume of the drop on
the reflector varies from 3 to 70µl.

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 Table 1. Thickness values and polymer mass deposited on the reflector to satisfy the thin film condition
for different energies

Drying metods
The behavior of drying polymeric samples is completely different compared with aqueous matrices.
The polymeric dispersion has a hydrophobic behavior on the silicon reflector, and as a consequence,
the film constrains the liquid drop to stay fixed on the reflector. This is due to the fact that polymer
dispersions have a very high elastic coefficient compared with the aqueous one, and for this reason,
the drop does not shrink on the reflector. In this way, during the drying process, the drop of the solvent
evaporates horizontally and not vertically; only a minute evaporation, a ring appears (22,23). Four
drying methods were investigaed in order to obtain the most homogenous thickness after drying: oven
at 40°C, vacuum, domestic microwave oven, and infrared lamp. The homogeneity was tested by
scanning electron microscopy (SEM). Figure 13 shows that homogeneous films were obtained when
drying processes are achieved under vacuum and microwave oven processes.

a) oven at 40°C c) microwave domestic oven

b) vacuum d) infrared lamp

Figure 13. Scanning electron photographs of polymeric thin films on silicon reflectors

3.- Applications
Impurities characterization
These polymers are usually expended in a solid state with 90% of purity. Traces of impurities may
modify their physicochemical properties so it is necessary to purify them in order to make confident
measurements. However, these processes are laborious because of the high viscosity of these colloidal
systems. For purification, dialysis, micro and ultrafiltration are usually employed and they are studied
further comparatively. Viscosity changes from diluted solutions (sols) to gels. It is necessary to adjust
a convenient method, according to the polymer concentration, for purification and for a simultaneous
performance control.

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The purification control is generally protein and iron quantification by using molecular and atomic
absorption techniques respectively. These methods, which are influenced by matrix effect, demand
sample preparation. In addition, the large amount of samples needed alter the system studied. The
TXRF in these systems shows two indispensable advantages: the small quantities of sample required
for the analysis and the avoidance of digesting the organic material. The rapid determination of
impurities makes it possible to carry out an immediate control of any step without altering the system.
The aim of this work was to put the determination of trace impurities by TXRF to use for the
performance study of three purification methods of polysaccharide systems.
Differents polymer sol and gel samples were directly analysed in order to identify the qualitative
characteristic of impurities. The impurities generally detected in the samples were Fe, Ca and S
(Figure14). A set of calibration standard solutions was made in MilliQ water, containing S, Ca and Fe
covering a range between 5 to 50µgL-1. Co was chosen as internal standard owing to its non-
interfering properties. The intensity of the Kα lines was measured for every element.
For measurements, an aliquot of 10µL of standard solution or sample was pipetted onto a quartz
glass sample carrier and allowed to dry to a thin film under an infrared lamp. Four replicates of each
sample were analysed.

1600

1400
Mo Incoh
1200

Mo Kα
1000
intensity (counts/s)

800

Si
600

400
Fe
Ca
200
Ar S

0
0 2 4 6 8 10 12 14 16 18 20
Energy (keV)

Figure 14. TXRF spectrum of a 1000 µg ml-1 scleroglucan sol sample before purifying it

Purification essays were carried out at 25°C.

Dialysis: 50ml of sol or gel to be purified were introduced into a dialyser tubing of a regenerated
cellulose membrane. The membrane is permeable to water and it permits passage of low molecular
weight compounds in aqueous solution while retaining materials with relative molecular weight 6000
and higher (exclusion pore 6000Da) (33). The tubing was immersed in a stirring container with milliQ
water periodically changed. A step of dialysis is the time needed to allow impurities to permeate
through the membrane and it states the moment to change the water in the container. It must be
determined for a given system by the analysis of the wastewater, which is changed when the
impurities are detected.
Samples of 500µL were taken, at different times, inside and outside the tubing, during the purification
process and they were deposited onto carriers and measured by the described procedure, after of 5µL
of standard solution (1000µgmL-1 Co) were added.
Relative analyte concentrations (relative to the initial analyte concentration) were plotted in Figure15
versus the corresponding step. Step 1, the original sample; steps 2, 3 and 4, the solution inside the
tubing after successive changes of external water, and step 5 is the final purified sample. The shape of

184
the curves was similar for both dispersions and gels but the time involved in each step was different.
The step times determined for the analysed systems result in a linear relationship with the polymer
concentration (Figure16):

tstep=a Cscl + b
where a = 0.0022h mL µg-1 and b = 2.08h
Cx/Ci

1
Ca
0,75

0,5 S
Fe
0,25

0
1 2 3 4 5
Step
Figure 15. Relative concentration (Cx/Ci) of the impurities during dialysis process of polymer sols and
gels. The concentration was plotted versus the step of the process

The process was finished when the concentration for all impurities was similar between two
successive steps.
Dialysis results effective after four steps for all analysed systems The S content was effectively
eliminated, but the final purified sample still contained 75% of the original Ca and 25% of the original
Fe impurities. As the membrane exclusion pore is 6000Da, it can be deduced that these elements are
very close to some big proteins and this method is useless to remove them.
step time (hours)

30
t step = 0,0022C scl + 2,08
25

20

15

10

0
0 2000 4000 6000 8000 10000 12000
Scleroglucan concentration (µ g ml-1)

Figure 16. The step times versus the polymer concentration for the dialysis purification of different
solutions

Ultrafiltration: The sample to be purified by ultrafiltration was put into a shaking 50mL container
while water under pressure was circulating through it and the filter. The filter was a membrane with
exclusion pores of 20000Da, to retain the macromolecules. Wastewater with small impurities was
expelled continuously at the exit.

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This method performance was investigated taking 500µL samples of wastewater and inside the
container, at different times, during the purification of 500 and 1000µgmL-1 polymer sols. Samples
were analysed following the procedure described above.
Figure17 shows the reduction of the impurities concentration inside the container during ultrafiltration
process of 1000µgmL-1 of scleroglucan solution. Relative concentration of the analytes (relative to the
initial analyte concentration) versus time was plotted. Results show that 30h are enough for the
purification of this sample; however 18h are necessary for a 500µgmL-1 polymer sample. It is
interesting to point out that, because of the blockage’s problems (plugging) of the membrane, it is
possible to purify only polymer samples below 1500µgmL-1 by this system. Polymer concentration
decreases with time because of the water flux. So, a compromise option between caused dilution and
obtained purity should be choosen.
Cx/Ci

1,5

1
Ca
0,5
Fe S
0
0 10 20 30

Time (hours)

Figure 17. The reduction of the analyte concentration during ultrafiltration process of a 1000µgmL-1
polymer solution. The analyte concentration relative to there at the initial time (Cx/Ci) in the container was
plotted versus the time

Figure18 shows results of the wastewater analysis, at different times, for the foregoing sample. The
major impurities concentration was found in about 5h, the free Ca and Fe went out quickly but the S
was slower. There may be equilibrium involved. After 30h, impurities were not detected, so the
process was stopped. However, approximately 50% of the original Ca and 25% of the original Fe
remain into the container. Proteins associated with these elements must be larger than the membrane
exclusion pores and do not pass through the filter.

12
Concentration (ng ml-1)

10
S
8

4 Fe Ca
2

0
0 5 10 15 20 25 30
Time (hours)

Figure 18. The analyte concentration (ngmL–1) in the wastewater versus the time during ultrafiltration
process of a 1000µgmL-1 scleroglucan sol

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Microfiltration: For microfiltration, a 50mL pump-syringe system was used. It consisted of a syringe
with a membrane filter (pore 12µm), which allowed macromolecules to pass through and retained the
largest particles, namely large proteins and bacteria.
The efficiency of this method was analysed by taking 500µL samples at the filtrate of a 4000µgmL-1
polymer solution and following the same described procedure for the measurement.
Microfiltration is a necessary process to eliminate big size impurities. It may be purified only polymer
solutions below 4000µgmL-1 by this method. The time spent to purify 50mL of 4000µgmL-1 polymer
solution was 8 hours. 25% of the original Fe, 75% of the original Ca and 100% of the original S were
found into the final purified sample (the filtrate) but the filter retained large particles with 75% of the
Fe and 25% of the Ca.
The minimum time consumption to achieve a maximum of efficiency, named the relative time, was
determined for the three studied methods in order to make an advisable comparison. It was referred to
a 50mL sample of 0.1% polymer sol, which could be purified by any of the three methods. Results are
shown in Table 2 together with several comparative features for the studied methods.

Cost Suitability for

polymer Relative
Technique time*
Equipment Running and Sol Gel (hours)
maintenance
Dialysis - Low very yes 15
Ultrafiltration High High yes (only up to no 4
0.15%)
Microfiltration Medium Medium yes (only up to no 5
0.4%)

Table 2. Important features comparison for the three studied purification methods
* Minimum time consumption to achieve a maximum of efficiency, relative to 50 ml of 0.1% polymer
solution.

It is interesting to point out that small size impurities are eliminated with dialysis and ultrafiltration
whereas the large ones are eliminated with microfiltration. The choice between these methods should
be based on the size of impurities, but they are generally used in a complementary way.
Ultrafiltration is an expensive method compared with dialysis and it provides similar results. However,
it is considerably useful in laboratories where the equipment can work continuously.
Dialysis is the most time consuming method but it is very suitable for both sol and gel polymer
solutions and it has the lowest costs. It is the recommended procedure for concentrated solutions and
gels. However, proteins will remain and perturb. Microfiltration followed by dialysis is recommended
to purify diluted solutions.
Control of chemical reactions
Classical polysaccharide detection techniques are complex and laborious, they have very high
detection limits, low sensitivity and sampling is generally required (24,25). This has been an
impediment to make fast and accurate experiences in both dynamic and static conditions. Labelling
with salts to detect the fluid by conductimetry has been used for years in spite of the problem caused
by differences in the mobility of the small ions and the large macromolecules (26, 29). Fluorescent
tagging by reductive amination of the aldehyde end group (30) is not appropriate for these polymers
because of their long chains. The substoichiometric addition of fluorophores and UV chromophores to
the hydroxyl groups has been reported (31). The radioisotope detection, a direct no-sampling

187
technique, has been used to study structures of compounds, reaction mechanisms and as a tool in
analytical chemistry, though the major application has been in biochemical research (32).
The generally most used tracers to label organic compounds are isotopic carbon and hydrogen. Other
important isotopes are those of nitrogen, oxygen and halogens. The method to incorporate an isotope
within an organic compound largely depends on the nature and availability of the tracer. The
introduction of the radioactive material should be at the latest possible stage of a multi-step synthesis,
avoiding handling as much as possible, and the recovery of the isotope from unwanted side-products
or unused starting material should be made without unnecessary dilution.
The reaction to label the polymer should be fast and simple and it should have an adequate
performance. The atoms should be fixed by a covalent union to assure that the detected signal
correspond to the macromolecule displacement. The working variables, namely pH, temperature and
time, should not increase the polymer degradation.
Iodation
The introduction of iodine atoms into the macromolecule makes the detection possible by any of the
above-mentioned two methods, depending on the iodine isotope used. The goal was to label the
macromolecule without altering its physicochemical properties (especially its transport properties);
this means the substitution of only a few OH groups per macromolecule.
In this work, an iodation in two steps is proposed to label the scleroglucan, following a procedure
previously reported for a-cyclodextrin (34). The first step or ‘activation’ consists of the introduction of
a very good leaving base (p-toluensulfonyl or p-tosyl) which is displaced by the iodide in the second
step, in situ (Figure19).

Figure 19. Reaction scheme for preparation of labelled scleroglucan

Tests were made by conductimetry, 13C NMR and TXRF analysis using the isotope 127I.
TXRF technique was adjusted for the analysis of scleroglucan labelled solutions (using 127I) and used
for test the evolution of the chemical reaction.
The covalent union and the location of the iodide atom into the scleroglucan macromolecule were
tested by 13C NMR.
The rheological behaviour was a method to test the polymer degradation during the chemical reaction
and to test differences between scleroglucan and the labelled product. The solutions of these
macromolecules are non-Newtonian fluids. Their flow curves, at a constant temperature, are not linear.

188
This means, the viscosity is not constant but depends on the shear rate in the fluid. The following
equation shows the relation used to characterize these fluids(34).

η = kγ& n −1
where, η is the viscosity, γ is the shear rate, k and n are constants for a particular fluid, k is a measure
of the consistence of the fluid and n is a measure of the degree of non-Newtonian behaviour (35).
The Gel-permeation chromatography (GPC) was a method to test the polymer degradation during the
chemical reaction and to test differences between scleroglucan and the labelled product.
The chemical reaction was tested both in acid and basic media due to the wide range of the polymer
triplex stability, from pH 3 to 12 (36).
In the first step, an esterification, a basic medium is indicated. The slowly dissolution of the p-
tosylchloride is produced while the reaction proceeds and the liberated hydrochloric acid is neutralized
by the addition of NaOH. The performance study of the reaction was followed by conductimetry and
pH measurements. Scleroglucan solutions have a very low conductance (Ω=10-6mS). The addition of
an electrolyte produces a drastic increment in this property. The advance of the chemical reaction can
be followed by the decrees in the concentration of the electrolyte reactant, via the conductance. This
method does not need any previous calibration study.
Figure 20 shows the recorded study of the reaction advance at two different temperatures. pH was
controlled to be between 7 and 11. During the first hour, evolution was not observed in the water–
scleroglucan-p-tosyl chloride system. The leaps of the conductance are due to the additions of NaOH,
until pH=11. While reaction produces H+, the conductance drops because of the neutralization. After
one hour, at pH=7, the conductance was caused by the produced NaCl and a new addition of NaOH
was made. An increase in the temperature produces an increase in the velocity of any reaction,
included the polymer degradation. The optimal temperature, time and pH (Table 3) were determined,
taking into account the limits imposed by the polymer degradation and the triplex destruction, that
could lead to some very different products.

NaOH (1M)
additions a
0,25

0,2 a
b
conductance (mS)

a
0,15 b
b

0,1

0,05

0
0 1 2 3 4 5 6 7
time (hours)

Figure 20: Conductance evolution during the 1st step of the labelling chemical reaction at two
different temperatures: a) 25°C and b) 60°C. Arrows mark the addition of same volumes of 1M
NaOH

189
 1st Step 2nd Step
Scleroglucan 0.2% 0.2%
concentration
pH 12 3
Temperature 50°C 80°C
Time 24 h 6h
Purification Dialysis (6000 Dialysis (6000
Da) Da)

Table 3: Optimal selected conditions for the chemical reaction

Before the second step, and only for testing, the first product (intermediate) was purified by dialysis
using 6000Da pore size membranes. The selection of the membranes was made considering the
blocking phenomenon and the purification time (with 14000Da pore size, blocking occurs in 24h, but
purification takes much more time) (15).
In the second step, the OH- competes in advantage (hydrolysis of the ester) against the iodide for the
displacement of the tosyl-attached group and the performance of the reaction drops. The acid medium
favours the displacement; the upper the efficiency is the lower pH.
The labelled scleroglucan was filtered and its purification by dialysis was tested by TXRF as it was
previously described.
The time, temperature and pH of the reaction were selected taking into account that the molecular
distribution and the viscosity of the product were as similar as possible to the original scleroglucan.
As indication of the influence of these variables on the final product, the properties of the products
obtained by five different conditions for the second step of the reaction are shown in Table 4.

Sample Relative Sample Detection Accuracy

volume Sensitivity destruction limits1
TXRF 10 µl 0,5-3 (to Co) yes 0.01% < 1%
Radioactivity No sampling 7 (to 131I) no 0.05% < 1%
1
for labelled scleroglucan solutions.
Table 4: Comparative characteristics for TXRF and Radioactivity analysis

In Figure21, RMN spectra from sclero, intermediate p-tosyl ester and iodide labelled product are
shown. Peaks were identified for scleroglucan (36-38), the tosyl group and the iodine group
respectively.
On the bases of this spectra and considering the RMN and X-Ray Diffraction studies on oriented fibers
and the conformational structure of scleroglucan triple helix (38, 36), we conclude that the substitution
was placed at the OH group of the C6, at the pendent β(1-6) linked glucose groups that protrude
outside from the triplex. A stereo view of the labelled product triplex is shown in Figure22.

190
 DMSO
Scleroglucan C-OH
C1 C5
C2 C4
C6
C3 -CH2I

Aromatic C
CH3-φ
C-H
-CH2-SO2-φ
C-SO2-
CH3-C

Figure 21: 13C NMR spectra of a) scleroglucan, b) p-tosyl ester and c) iodine labeled scleroglucan

Quantitative TXRF analysis showed that one p-tosyl group kept attached by every one monomer unit
of the polysaccharide in the first step of the reaction. In the second step, a 60% of the introduced tosyl
groups were substituted by iodide and the rest of them were hydrolysed. In Figure 23 the TXRF
spectra from the three macromolecules are shown. The fluorescence energy characteristic provided by
S and I atoms permitted their identification.
The radioactive performance was measured. A comparison between the activity of the original Na131I
and the lost activity during the dialysis purification showed that the 131I isotopes were effectively
attached to the macromolecule. This concludes in a 15% radioactive performance for the reaction.

Figure 22: Stereo view of the triplex of iodine labeled scleroglucan. C and O atoms are grey and
white circles respectively. The two iodine atoms corresponding to this portion of the
macromolecule are indicated with white bigger circles. H atoms are omitted

191
 Si Co

1400
I Iodide
Ar scleroglucan
1200

Mo
1000
Intensity (counts)

800
Tosyl
S scleroglucan

600

400

200
Scleroglucan

0
1 3 5 7 9 11 13 15 17 19 21
Energy(keV)

Figure 23: TXRF spectra from the three macromolecules. Kα and Lα lines from S and I atoms are
indicated

Comparison between scleroglucan and the labelled product

Rheological measurements were done in order to determine the change in the macromolecule
behaviour caused by the chemical reaction. In Figure 24, the log-plots of viscosity (η) and shear rate
(γ’) measured at 25°C on samples of the same concentration of scleroglucan and iodide derivatives
show coincident exponent. It result in n = 0,72 for 0.2% of macromolecule concentration. The p-tosyl
ester showed an exponent slightly smaller (n = 0.70).

10000
viscosity (Pa.s)

1000

100

10

1
0,1 1 10 100
shear rate (s -1)

Figure 24. Viscosity (Pa. s) and shear rate (s-1) measurements plot for (•) scleroglucan, (‹) iodine
labeled scleroglucan and (¯) p-tosyl ester derivative. The three different macromolecules fit in the same
equation η= 240 γ’ –n. With n=0.72 for the first two and n=0.70 for the p-tosyl ester

192
 The aproximate weight-average molecular weights of the samples listed in Table 5 were obtained
using the Mark-Houwink equation.

[η ] = kM a
where M is the mass of the polymer molecule, k is a constant determined by measuring M for several
homologues with different mass of the same polymer and a is the Mark-Houwink exponent related to
the shape of the molecules. The value of k = 4. 10-4 according to previous results (35-38) was used for
calculations. The intrinsic viscosity determined for scleroglucan triplex solutions was [η]= 113cm3/g.
These values allow estimating the corresponding Mark-Houwink exponent value for these systems: a
= 0.8 (39).
A second value to estimate the weight-average molecular weights of the samples was obtained by the
exclusion profile by gel permeation chromatography. The polymers showed narrow molecular weight
distributions and the data according to the calibration curve with standard dextrans were listed in
Table 5. The estimated Mw values were in agreement with the results of intrinsic viscosity
determinations.

n MW1 MW2 Mw/Mn1 [η ] Water DMSO

Molecular solubility solubility
Weight- Weight- Intrinsic
Distribution
Rheologica average average viscosity
(Polidispersit
l exponent molecular molecular (cm3/g)
y)
weight (Da) weight (Da)
Sclerogluca 0.72 4.0 106 3.9 106 1.20 113 very very
n
Tosyl 0.70 5.1 106 5.3 106 1.35 140 difficult very
sclerogluca
n
Iodide 0.72 4.2 106 4.1 106 1.23 120 very very
sclerogluca
n
1
GPC mesurements.
2
Viscosity measurements
Table 5. Comparative physical properties of 0.2% scleroglucan and derivatives solutions

There were no significant differences between the scleroglucan and the labelled iodide macromolecule
in the measured properties. Rather different was the case of p-tosyl ester due to the weight and number
of the p-toluensulfonic groups. Hydrolysis of these groups is very important in the second step of this
method, then the final labelled macromolecule is much more similar to the original that the
intermediate one.
The iodide labelled scleroglucan showed in its molecular structure two iodine atoms each five
monomer units. It make a difference of 5% in weight but the transport behaviour was very similar to
the original scleroglucan. Replacing it with that detectable macromolecule makes possible the
expansion of the dynamic experiences to non-transparent porous media.
We consider successful the result of the chosen procedure to label the scleroglucan macromolecule in
a safe way.

193
Comparison between the techniques for detection
Comparative characteristics belonging to the employed techniques are showed in Table 6. Their
detection limits and accuracy are very similar. Both techniques allowed detection and quantification of
scleroglucan solution in concentrations from 10-2 %.

Sample Relative Sample Detection Accuracy

volume Sensitivity destruction limits1
TXRF 10 µl 0,5-3 (to Co) yes 0.01% < 1%
Radioactivity No sampling 7 (to 131I) no 0.05% < 1%
1
for labelled scleroglucan solutions.
Table 6. Comparative characteristics of TXRF and Radioactivity analysis.

Even though the p-tosyl ester can be detected by TXRF, the reaction should proceed to the second step
to avoid difficulties originated by the small differences in the behaviour of the macromolecule.
The proposed techniques for detection are complementary. Their usefulness depends on the condition
of the experience.
Adsorption onto glass
Adsorption is a surface physico-chemical phenomenon caused by the interaction between a fluid’s
molecules and a solid’s molecules. When the fluid is a solution, the amount of solute adsorbed is
proportional to the number of active sites on the solid’s available surface, and depends on the
concentration of the solution and the forces of interaction between the active sites and the substance’s
molecules. These interactions are related to the size and spatial conformation of the molecule of solute
as well as to the structure and chemical nature of the solid (40).
The mechanism through which this phenomenon takes place is not certain yet and is not easy to study.
The analysis is even more complex when it comes to polymeric dispersions, although there are
models, most of them statistical, which try to provide an explanation for some well-known
experimental results (41).
The polymer selected for this work was scleroglucan (13) and as solid adsorbent, glass microspheres
were used, being this the material commonly employed in laboratories to simulate porous media and
filters. The objective was to study the adsorption of aqueous dispersions of scleroglucan of different
concentrations on a glass surface. In order to do so, the isotherm of adsorption was experimentally
obtained. The isotherm of adsorption supplies information regarding the mass of solute adsorbed per
mass unit of adsorbent (Mad) as a function of solution concentration (Csc), once reached the
equilibrium state.
The macromolecule used in this work had been previously marked with iodide atoms (42). Iodide
concentration before and after the interaction was measured by Total Reflection X-Ray Fluorescence
(TXRF). Adsorbed polymer films were evaluated as given by concentration measurements.
Some general equations of adsorption that relate Mad to Csc are:
Langmuir:
aC sc
M ad =
k + C sc
and Freundlich:

M ad = kC scn

194
being the first one usually adequate to describe gas-solid systems and the second to liquid-solid
systems. The adjustment parameters: a, k and n are characteristic for each material (39).
Figure25 shows a graphic representation of these and other common equations.
Aqueous solutions of iodide labelled scleroglucan and Si-Na-Ca glass microspheres with 400-600µm
diameter, 80% sphericity, 1.5-1.6g.cm3 apparent density and 3.7cm2g-1 specific surface area were used
in this work. An aliquot of the dispersion of the chemically labeled polymer is deposited on a
reflecting surface and dried to a thin solid film.

Freundlich (liquids & solutions)

Mad
Langmuir (gases)

Lineal (aproximation
for some simple
systems)

Freundlich(liquids & solutions)

unfavorable adsorption

Csc
Figure 25. Characteristic isotherms for different systems

Adsorption curves were obtained measuring polymer concentration before and after different time
intervals of contact, for a 1:3 macromolecule-glass ratio. Initial concentrations ranged between 30 and
1000mgL-1.
Ten samples of each concentration of scleroglucan studied were prepared as follows:
-A measured volume of labeled scleroglucan solution was placed together with a weighed amount of
glass microspheres in a cylindrical cell and treated in a thermostatized (25°C) rotatory stirrer at 6rpm.
-At certain time intervals, one of the samples was extracted for analysis. The microspheres were
separated from the scleroglucan aqueous dispersion and scleroglucan concentration was determined by
TXRF spectrometry as iodide concentration.
The adsorption isotherm was generated based on these experimental data.
Figure 26 displays the adsorption curves obtained for the aqueous dispersions studied. The time
required to reach the equilibrium state is indicated on the graphic.

195
 0,16

Mad(µgpoly.gsph-1)
0.1 mgL-1
0,14

0,12 Time needed to reach

equilibrium state.
0,1
0.05 mgL-1
0,08

0,06

0,04
0.01 mgL-1
0,02
0.003 mgL-1
0
5 25 45 65 85
Time(min)

Figure 26. Experimentally obtained adsorption curves. Polymer concentration as a function of time for
different initial concentration

Figure 27 shows the adsorption isotherm obtained with base on the adsorption curves.
A large number of configurations at the solid-solution interface are possible. It takes several
parameters to describe the state of a polymer at the interface, which makes it difficult to confirm or
deny the various theoretical models found in bibliography. Considering the sophistication of many of
those models, it is interesting to notice that our polymer adsorption data fit the simple Freundlich
equation, within experimental error.
The adjustment parameters n and k were calculated to fit the experimental data:
kexp = 578 and nexp=2,93.
Mad (µgpoly.gsph-1)

90

80

70

60

50

40

30

20

10

0
0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5
Ceq poly (%)

Figure 27. Experimental adsorption isotherm (25ºC) for scleroglucan solution on glass

196
Polymer layer formation is unavoidable when even weakly attractive surfaces come into contact with a
polymer solution. Even for extremely dilute polymer solutions, polymer layers develop with densities
that may be many orders of magnitude larger than the bulk polymer concentration. However, the
amount of polymer retained per gram of adsorbent in these experiments is quite small, as can be seen
in Figure 26. This led us to propose that there is a competition between the water molecules and the
macromolecules for the surface active sites. Therefore, only a slim layer of polymer could be weakly
adsorbed on the glass.
Experimental results show Freundlich dependence for the concentrations of polymer studied. The time
to reach the equilibrium state increases with the scleroglucan concentration.
Though TXRF has been rarely employed to analyze polymeric systems, we have found it a very useful
technique to study trace adsorption in viscous aqueous solutions. Its high sensibility allows working
with diluted Scleroglucan solutions. An advantage to point out is the direct measurement of the
sample, which ensures the preservation of the polymer structure and minimizes the risk of
contamination.
Ionic Diffusion
Transport properties in macromolecular systems have applications in a wide variety of industrial fields
ranging from drug controlled release or diffusion-controlled kinetic chemical processes to Enhanced
Oil Recovery (13). To design technological devices, it is necessary to have an understanding of the
transport properties of the diffusing species in a given matrix (polymer-solvent) which are influenced
by changes in structural characteristics (39). This work presents a systematic study of the diffusion
behaviour of nitrate salts of Ba and Mn through aqueous dispersions of Scleroglucan and
Polyoxyethylene (POE) polymers, respectively, in 0,01 to 1% concentration ranges. The evaluation of
the metal ions concentration through both polymeric matrices and pure water is carried out by TXRF
analysis. This microanalytical technique is especially adequate for this purpose considering that the
capillary method selected for the experiments have the major precision when the minor amount of
specimen is used. The proposed TXRF methodology is a safety option replacing the traditional
radiotracer determinations and it has not been reported previously by other investigators. In this case
the need of an internal standard addition, which is the customary methodology in TXRF
quantification, is avoided by using the scattered radiation method (43).
Polymers used in this work were Scleroglucan and POE
In this work, the capillary method was employed for determining diffusion coefficients. This method
is only applicable when a very small amount of the diffusion agent is measured (41). The diffusion cell
consists of a precision-bore capillary tube, 5cm long and 0.05cm diameter. Once capillaries were filled
with a given polymer-salt system, they were sealed shut on the bottom by slightly heat and immersed
into a large bath. The initial salt concentration was 50µgL-1 for all the experiments. During the
experiment, the Mn or Ba nitrates diffuse outside of the capillaries because of the concentration
gradient, the slower and viscous POE or Scleroglucan macromolecules remain inside. We have not
observed in our experiments, by means qualitative chemical essays using phenol-sulfuric reaction (25),
the leaching out of the polymer itself from the capillaries. The purpose of each experiment was to
obtain the concentration profile (the concentration as a function of the time) for an ion (Ba or Mn)
diffusing through a polymer solution. For each experiment, a set of 10 capillaries, containing the same
salt and polymer concentrations, was put inside a one liter, stirred, thermostated (25 ºC) aqueous bath.
Figure 28 shows a scheme of the experimental setup. Each 30 minutes time, one of the capillaries was
removed from the bath (one point of the concentration profile) and its content (c.a. 9.8µL) was
deposited onto a reflector. These samples were dried in a microwave oven at low power. Finally, Ba or
Mn concentrations were determined by TXRF using the Compton peak as internal standard. The
measuring time was 500s.
For instance, 20 concentration profiles (10 points each) for BaNO3 in different concentration solutions
of POE were obtained from 20 experiments of 10 capillaries each.

197
 Water bath (25ºC)

STIRRER

Figure 28. Scheme showing capillaries filled with polymeric solutions (Scleroglucan or POE) which
contain Ba or Mn nitrates salts immersed into a water bath (25ºC). The Mn or Ba nitrates (z) diffuse
outside of the capillaries because of the concentration gradient, the slower and viscous POE or
Scleroglucan macromolecules ( ) remain inside during the experiments

Sensitivities for Ba and Mn in these polymer systems were determined by the corresponding TXRF
calibration curves between 10 to 50µgL-1 concentration ranges. Obtained values were 0.53 and 3.02L-
1
µg, respectively. The metal ion diffusion coefficients (D) through the polymeric matrices were
obtained by fitting the concentration profiles obtained (salt concentration vs. time) with the
corresponding solution of the Fick Law (44).
⎛ ⎞
2 ⎛⎜ 2 ⎜⎜⎜ 2 ⎟⎟
−π 2n − 1
⎞
⎟
⎜ ⎟ ⎜
Dt 4l ⎟
⎟
⎞ ∞ ⎛
⎝ ⎠
⎛
⎟ ∑ ⎜ 1 (2 n − 1) 2 ⎞⎟e
2 ⎜⎜ ⎟⎟

C C = ⎜⎜ 8 π
⎝ ⎠

⎟
t 0 ⎝ ⎠n = 1 ⎝ ⎠

Where, l is the length of the capillary, t is the diffusion time and n is serial number determined by the
numerical method, Ct and C0 are the average concentration, determined by TXRF, present in the
capillary at times t and zero, respectively,.
By using this numerical method, the precision in D determinations were better than 0.3%. (This value
does not take into account the precision in C determinations and it is generated by the computer
calculation).
For the discussion of the results, it was used a comparative D/D0 (where Do is the diffusion coefficient
found in pure water.).
Figure 29 shows the diffusion behaviour experimentally determined for Mn and Ba salts in different
POE and Scleroglucan concentration matrices. The diffusion coefficients, obtained by fitting the
experimental salt concentration profile into each polymer system (different polymer concentrations) to
the solution of the Fick equation, were related to the diffusion coefficient obtained by the same
processes in pure water. These relative diffusion coefficients (D/D0) vs. the polymer concentration
were plotted for Scleroglucan and POE, separately.

198
 1.6
Transition zones
D/Do
1.4

1.2

1.0

0.8

Ba - sclero
0.6
Mn - poe
0.4 Mn - sclero
Ba - poe
0.2 Semi-diluted
Diluted
solutions solutions Gels
0.0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
% polymer
Figure 29. Relative diffusion coefficients (D/Do) experimentally obtained for Mn2+ and Ba2+ ions through
different concentrations of Scleroglucan and POE solutions

For these polymer solutions the well-defined concentration regimes (45,46), experimentally
determined, gave transition ranges between diluted and semi-diluted systems environs 0.1% for
scleroglucan and 0.2% for POE. The transition ranges to concentrated solutions or gels was close to
0.6% for scleroglucan and 0.8% for POE.
Changes in the diffusional behaviour of the ions are in coincidence with changes of the concentration
regimes (Figure 29) confirming that transport through these systems is affected by the structural
modifications on the matrix.
These results are in agreement with similar studies made by our research group using independent
methodologies such as free diffusion with UV spectrometry detection and Stokes cells with
conductimetry detection (47).
There are several physical models to explain these behaviours in the different concentration regimes.
In the diluted regime, the diffusion coefficients of studied ions decrease when the concentration
increases. These small ions show hydrodynamic obstruction diffusion behaviour. The model of Cukier

−κ C( )1 2
D D0 = e
(48) was used in this concentration zone:
where the parameter κ depends on the size of the diffusing specie.
In the semi-diluted regime, the diffusion coefficients surprisingly increase with the polymer
concentration. The size of the clusters increases whereas their number decreases (when polymer
concentration increases). The possibility of ionic channels self-generated can explain the highest
experimental diffusion coefficients (49), but this behaviour has not been previously observed and there
is not a physical model to apply. Further studies are undertaken on this topic.
In the concentrated regime (gel), the diffusion coefficients diminish with the increase of polymer
concentration. This diffusional behaviour is determined by the size of both the ions and the polymer

199
matrix. Tortuosity is the dominant interaction (50). The model of Phillies (51)] was used in this
concentration zone:

ν
−αC
D D0 = e
where α and ν are parameters that depends on the size of the diffusing specie and the polymer
matrix, respectively.
It has been shown that the transport through these systems is affected by the structural characteristics
of the system because changes in the diffusion behaviour of solutes are in coincidence with the
concentration regimes of the matrix. Charge, shape, molecular size, and the structural complexity of
the matrix clearly affect the diffusion properties of species. The different hydrodynamic radii explain
the diffusional behaviour founded for nitrate salts of Ba and Mn that have the same charge. TXRF is a
very suitable technique for the study of these high viscosity systems considering the small amount of
sample available from the capillary method.
Analysis of Compton classification
In polymeric samples, the dark matrix is made up of carbon, oxygen and hydrogen; because of its low
average atomic number, it does not contribute to the detected fluorescence, but gives rise to Compton
and Rayleigh scattering (52). Total reflection of the primary beam eliminates the high background due
to scattering from the sample substrate support material, but obviously not the scattering originated in
the sample itself. In general, the useful information of a typical X-ray spectrum is restricted to the
region of characteristic lines. In this region, a series of discrete X-ray spectral lines—characteristic of
the emitting element and having various relative intensities—can be observed; it is located on the
lower energy side of the spectrum. The information coming from the neighborhood of the exciting
lines is not frequently used (53). The aim of this work is to use information present in the spectral
region corresponding to elastic and inelastic scattering of the incident X-rays, obtained under total
reflection conditions, combined with chemometric techniques, to classify synthetic and natural
polymers having large molecular weight. Chemometric techniques (54,55) are concerned with formal
methods for the selection and optimization of analytical methods and procedures and for the
interpretation of data. Statistical treatment, with the aim of grouping and classifying the elements of a
population, constitutes the main objective of numerical taxonomy. In this sense, it is a complementary
tool for a better understanding of analytical results.
For each polymer, triplicate solutions having three different concentrations were made by dissolving
the solid polymer (e.g. 0.2, 0.4 and 0.6g) in 100mL high-purity water. The solutions were stored for a
maximum of seven days under continuous low stirring in order to avoid mycrogel formation.
For measurements, an aliquot of 10 µl of each final solution was pipetted onto a quartz glass sample
carrier and allowed to dry to a thin film under an infrared lamp. Four replicates of each sample were
analyzed. To improve the statistical relevance of the data, 20 samples from each batch were measured
and the results were averaged. All the spectra are normalized to allow comparison.
Before attempting this classification using a clustering algorithm (56), the data were evaluated using
principal component analysis (PCA); once the principal components were identified, we proceeded
with the clustering technique (57-59).
The MATLAB™ software was selected for both chemometric tools. The PCA analysis, a
recommended visualization technique, reduces the redundant original variable space to an optimal
lower dimensional abstract space, whose variables contain uncorrelated information. This dimensional
reduction allows the objects to be mapped in a two or three-dimensional space (biplot). The
coordinates of objects in this new abstract space, the so-called scores, can be used as input data for the
clustering analysis; each one of these objects has an associated variance, indicating the relative weight
of the variable in the classification scheme.

200
Figure 30 shows spectra obtained from three different polymer solutions. It can be noted that the
Rayleigh and Compton peaks depend on the matrix composition. The Rayleigh peak is observed
between 17.35 and 17.46 keV and the Compton peak from about 16.79 to 16.97 keV.

Figure 30. Incoherent and coherent scattering regions of xanthan, scleroglucan and 2,3,6-tri-o-
ethylamylose TXRF spectra

The cumulative contributions to the total variance of the first two resulting principal components (PC1
and PC2) are 95.53 and 0.88%, respectively.
Figure 31 shows a biplot of the normalized scores in PC1–PC2 space. From this plot one can see that it
seems reasonable to attempt a clustering of these objects in terms of these PC’s.
The clustering technique is based on the similarity of the samples expressed according to a coefficient
of dissimilarity.

201
Figure 31. Results from principal component analysis of polymers. (+): loads of the original variables. ( ):
scores of the samples

Figure 32 shows a dendrogram obtained using two PC, autoscaled data, Mahalanobis distance and the
k-nearest neighbors algorithm. This type of diagram constitutes a useful representation that facilitates
the comparison of samples with large concentration ranges for the various components. Six distinct
clusters are present; five of them have a high degree of similarity, and the remaining one is associated
very loosely. Each of the five groups with high similarity corresponds to a well-defined family of
polymers: scleroglucan, xanthan, POE, glucomannan and 2,3,6, tri-o-ethylamylose. The sixth cluster
contains PAM polymers. It must be noted that clustering is insensitive to concentration in the range
studied; in fact, the dispersion of the points due to concentrations is very similar to that due to
experimental error. It is interesting to note that while PAM polymers contain nitrogen atoms, the other
compounds are made up only of C, O and H.

Figure 32. Dendrogram obtained from the PC model data usingk-means nearest and Mahalanobis
distance

The fact that each polymer have some energies scattered with great intensity variations allowed us to
classify them. In order to detect more information coming from the matrix of data (intensity vs energy)
the energy Compton range was studied for each polymer.

202
As example, Figure 33 shows the intensity energies standard deviation plot obtained for the
scleroglucan dispersions. It can be noted that for some variables (energies) the standard deviation
presents maximum values, the rest of the energy values contain correlated information and this is not
relevant for classification.

0.07

0.06
Standard Deviation of S am ples

0.05

0.04

0.03

0.02

0.01

0
0 20 40 60 80 100 120 140
V ariable

Figure 33. Intensity energies standard deviation in the Compton range for scleroglucan samples

In order to reduce the number of variables, PCA was achieved. Figure 34 shows the plot of the
loadings from the former matrix of spectra energies for the two first PCs. It can be seen which are the
detected energies and their location in the PC space as well as the location of the characteristic
energies that make samples of same polymers appear together. Most of former variables are correlated
and have no useful information (values are indicated in keV).
Chemometric tools are useful to extract information from the dark matrix contained in the Compton
region during classical TXRF analysis of polymers. The characterization of the polymers was possible
and their identification and quantification could be planned with adequate and calibrated software,
taking into account that chemometric tools are nowadays integrated with spectroscopic
instrumentation. The proposed methodology is fast and no sample preparation is required with the
advantages of preventing changes in the chemical structure of the polymers or possible contamination.
Compton region was selected because molecular polymer structures differ above all in outer electrons.
Finally, we are aware that to explain these experimental facts a theoretical study should be developed
to correlate the characteristic energies with the molecular structure of the polymers.

203
 1
15.82KeV

0.5 15.44KeV
16.3KeV
16.48KeV
15.76KeV
15.96KeV 15.3KeV
0

16.16KeV
150
100
-0 . 5 50
1 0.5 0
0 -0 . 5
V a ria b le

Figure 34. Loadings of the intensity energies plot for the scleroglucan samples

Estimation of carbon plus oxygen content in polymers

The information about the matrix contained in the scattered X-rays has been exploited in several ways.
Their intensities depend on the photon energy and the sample substance, that is the mean atomic
number Table 7. Coherent (Icoh) and incoherent (Iin) scattered radiation give information about the
sample itself. With the aim to investigate this idea, it was observed that a linear relationship is
obtained by plotting the Is=Icoh/Iin vs. %of C+O for each polymer. This can be explained regarding that, in
this particular case, the only three constituents of the polymers are C, O and H. C differs from O by
only two atomic numbers. It can be assumed that these elements have similar scattering properties and
x%of C and y%of O produce the same intensity as x+y%of C. That it is sufficient to consider the carbon
plus oxygen as if these elements were an equivalent weight of carbon.
This is a further advantage of this method. This additional information can be obtained without any
additional measurement. Only the intensity of the coherent peak must be included when the spectrum
is fitted. It must be understood that this estimation is not very exact on account of the assumptions that
were made. However, this information is not time consuming and avoids the laborious analytical
determination of C plus O concentration. Once the calibration is completed using known polymers, the
estimation of C plus O content in a new one can be determined by direct interpolation in the
calibration curve.

Table 7. Relationship between Is and concentration of (C +O)g /100 g in polymers

The method for estimating the content of C+O is much more rapid than the usual analytical methods
and gives additional information about the matrix, simultaneously with trace element determinations.

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Susana Boeykens
LaQuiSiHe (Laboratorio de química de Sistemas Heterogéneos)
Facultad de Ingeniería. Universidad de Buenos Aires
Av. Paseo Colón 850. 5to Piso. C1063ACV. Buenos Aires, Argentina
sboeyke@fi.uba.ar

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