A comparison study of analytical perform

Accepted Manuscript
A comparison study of analytical performance of chromium
speciation methods
Posta József, Nagy Dávid, Kapitány Sándor, Béni Áron
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DOI:
Reference:
S0026-265X(19)30372-8
https://doi.org/10.1016/j.microc.2019.05.058
MICROC 3958
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Microchemical Journal
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23 May 2019
Please cite this article as: P. József, N. Dávid, K. Sándor, et al., A comparison study
of analytical performance of chromium speciation methods, Microchemical Journal,
https://doi.org/10.1016/j.microc.2019.05.058
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ACCEPTED MANUSCRIPT
A comparison study of analytical performance of chromium speciation
methods
Posta József a*, Nagy Dávid b, Kapitány Sándor c, Béni Áron d
Department of Landscape Protection and Environmental Geography, Faculty of Natural
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a
Science and Technology, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
Department of Mineralogy and Geology, Faculty of Natural Science and Technology,
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b
c
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University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
Institute of Environmental Sciences, University of Nyíregyháza, Sóstói út 31/B, 4400
Institute of Agricultural Chemistry and Soil Science, Faculty of Agriculture and Food
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d
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Nyíregyháza, Hungary
Science and Environmental Management, University of Debrecen, H-4032 Debrecen,
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Böszörményi út 138.
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* Corresponding author: e-mail: posta.jozsef@science.unideb.hu
Running title: A comparison study of analytical performance of chromium speciation methods
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Keywords: Speciation, Chromium, Extraction, AAS
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Abstract
Our research group have been contributing to the development of speciation analytical
techniques with the elaboration of various chromium speciation methods since 1990. In the past
period 20 divers techniques have been developed for the separation, enrichment and
determination of toxic chromium (VI) and essential chromium (III) forms in environmental
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samples. The aim of present work is to compare the analytical performance of introduced
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chromium speciation techniques, and to get an overall picture about the limit of detection and
range of quantification of the on-line and off-line techniques, and to present the optimal
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experimental arrangements to achieve the best possible analytical performances using the latest
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analytical tools. The analytical performance of on-line chromium speciation techniques can be
improved by the appropriate enrichment of chromium forms, by using interface connecting the
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separation/enrichment unit and the element selective detector with the highest sample
introduction efficiency, and by using a detector with the best possible sensitivity. The lowest
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limit of detection (3σ) for Cr (VI) (20 pg/mL) could be achieved by using C-18 chromatographic
separation/enrichment column, hydraulic high pressure nebulizer (HHPN) and acetylene –
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nitrous oxide flame emission detection unit. The10 pg/mL (3σ) limit of detection of off-line
chromium speciation method was reached by 50-fold enrichment using liquid-liquid extraction
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(LLE) and graphite furnace atomic absorption spectrometry (GFAAS). These techniques are
suitable for the chromium speciation analysis of natural samples with various matrices.
Keywords: Speciation, Chromium, Extraction, Preconcentration, AAS
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Introduction
The development of elemental speciation techniques has been a great challenge for analytical
chemistry at the end of 20th century and at the beginning of 21th century. The aim in speciation
analytics is to determine not only the total concentration of an element, but the concentration
of individual species with different oxidation states and bonding environment, respectively. It
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is of vital importance, since the species of an element are responsible for the toxicity and
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physiological effects, not the total concentration of the element [1-3]. This new field of
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elemental analytics is called in general speciation analytics. The number of publications has
been increased significantly in the last 30 years in this field of science that shows the importance
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of this topic in different areas of life.
Chromium is a good example for the difference in physiological properties of species of an
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element. Chromium occurs in nature in two relatively stable oxidation state, as chromium (III)
and as chromium (VI). The biological, physiological effect of these species are completely
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different. Each existing species of toxic elements like arsenic, mercury, cadmium and lead are
toxic for some extent, however, in case of chromium, chromium (III) is essential for living
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organisms, while chromium (VI) is toxic, carcinogenic. Chromium (III) plays an important role
in metabolic processes by enhancing the activity of certain enzymes and stimulate the synthesis
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of cholesterol and fatty acids [4]. Chromium (III) can be found as an ingredient in some
commercially available complex compounds, medicines, medicinal products (like. chromium
pycolinate pills, Centrum multivitamin products, Béres-drops, etc.)
The contradictory physiological effect of chromium (III) and chromium (VI) had demanded the
development of such analytical techniques that makes it possible to determine the concentration
of both species in natural samples (drinking water, surface water, sea water, blood serum, food
products, medicinal products, etc.), respectively.
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Our research group at the Department of Inorganic and Analytical Chemistry, University of
Debrecen has been contributing for the development of chromium speciation techniques in form
of international co-operations since 1992. In our laboratory several techniques have been
developed, which are capable for the on-line and off-line determination of chromium species in
a wide range of samples, whereas some of them are under further developments. This study
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aims to describe the working principal of the developed 20 different chromium speciation
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techniques and to compare their analytical performances.
The speciation analytical methods are generally coupled techniques, since they often require
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more than one analytical system to be used. Speciation techniques can be categorized based on
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their implementation as on-line or off-line techniques. On-line speciation arrangement means
that the sample liquid is directly analyzed in the element selective detector after the preceding
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separation/enrichment of the species of the studied element. As a result the analytical response
signal appears on the display in a few seconds after the start of the analysis. The separation and
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enrichment of chemical species are isolated in time and space from the analytical detection, in
case of off-line techniques. Off-line techniques are established on the one hand when the direct
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and continuous coupling of the detector with the chromatographic system is not executable. An
example is the continuous fractionation of eluted solution from a chromatographic column. On
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the other hand, some analytical detection systems, like graphite furnace atomic absorption
spectrometry (GFAAS) are not suitable for the on-line measurements due to the periodical
operation of the instrument.
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1. On-line separation/enrichment techniques
The experimental arrangement of on-line elemental speciation analytical techniques has three
major blocks:
1. Separation/enrichment system that separates the species of the studied element from
each other, and if possible enriches one of the separated species.
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2. Interface is the sampling device that couples the separation/enrichment system with the
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element-selective detector.
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3. Element-selective detector that performed the qualitative and quantitative analysis of
the separated species.
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During the development of the different methods the main focus was on the achievement of
best selectivity, separation, enrichment and limit of detection with the highest analytical
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performance.
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1.1.On-line separation of chromium (III) / chromium (VI) using C-18 column [5]
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Cr (III) is present in nature in form of Cr3+ ion and aqua complex, while the dominant forms of
Cr (VI) are CrO42- and Cr2O72- anions. Thus the chemical behavior of Cr (III) and Cr (VI) is
different towards alkyl ammonium salts (TBA-salts). TBA salts do not react with Cr (III), while
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ion pair complexes may form with the negatively charged Cr (VI) species. These ion pair
complexes are retained on C-18 columns due to the hydrophobic interaction between the
stationary phase and the alkyl chains of the complex. In aqueous medium the Cr (VI) – TBA
complex is entirely bound to the C-18 column. The strength of hydrophobic interaction can be
reduced by increasing the concentration of methanol in the eluent. At 30% (v/v) methanol
concentration, the separation of the 2 species is so that after the Cr (III) leaves the detector, the
Cr (VI) immediately follows it. Under these condition the speciation analysis is carried out in
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less than 30 seconds, which analysis time is outstanding among the other published methods in
the literature.
1.2. Enrichment of Cr (VI) using TBA salt with C-18 column
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The Cr (VI) – TBA complex is completely retained on the C-18 column, if the eluent is
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methanol-free aqueous solution. The bound complex can be eluted from the column effectively
and quantitatively, using methanol. This observation was employed for the enrichment of Cr
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(VI). The larger volume of Cr (VI) containing solution is the passed-through the C-18 column,
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the more efficient the enrichment is. After the enrichment the eluted Cr (VI) is flushed to the
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element-selective detector (FES, FAAS, ICP-AES, ICP-MS).
1.3. Separation of Cr (III) – Cr (VI) using potassium hydrogen phthalate [6, 7]
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When a solution containing Cr (III), Cr (VI) and potassium hydrogen phthalate is passed
through a reversed phase C-18 column, Cr (VI) passes through the column without retention,
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on the other hand Cr (III) is quantitatively bound to the column in form of a phtalate complex,
which can be completely eluted in a subsequent step using methanol. The retention of Cr (III)
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on C-18 reversed phase column has not been reported earlier. A possible reason for the
separation is the development of nonpolar interaction between the filling material of C-18
reversed phase column and phtalate. It has been not cleared yet which forces are awakening
between the Cr (III) and phtalate during the sorption. Further investigations are needed to
answer the above mentioned question, especially with respect to the reason of high selectivity
of phtalate to Cr (III) compared to other cations.
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1.4. On-line enrichment of Cr (III) using potassium hydrogen phthalate
Cr (III) is retained in the reversed-phase C-18 column in the presence of KH-phtalate until it is
eluted with methanol. It is possible to use even larger sample volumes (e.g. 5 mL) for Cr (III)
speciation with KH-phtalate than the conventional sample volumes (100 µL) in
chromatography. A 50-fold enrichment can be achieved in the above example after the Cr (III)
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is eluted from the C-18 column with methanol.
1.5. Enrichment of Cr (VI) using APDC in a sorption loop [8]
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An on-line sorption technique has been adopted to compensate the limited application of C-18
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column in chromium separation [9]. The working principle of the sorption method is the
formation of poor solubility complex between Cr (VI) and ammonium pyrrolidin
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dithiocarbamate (APDC) that is quantitatively absorbed on the wall of plastic capillary through
which the solution is flowing through. The material of the plastic loop is PEEK (polyether-ether
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ketone), commonly used in HPLC techniques. The sorbed Cr-PDC complex can easily be eluted
from the inner wall of capillary with isobutyl-methyl-ketone (IBMK) and consequently flushed
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into an element-selective detector. This sorption technique can be applied for the enrichment of
chromium (VI) in case of samples with high salt and organic material content that would
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damage the C-18 column.
1.6. Separation of chromium species with capillary electrophoresis (CE-ICP/MS) [10, 11]
The difficulty of separation of the chromium species using capillary electrophoresis arises from
the different electrophoretic properties of the two species. The Cr (III) species, like Cr3+,
Cr(OH)2+, Cr(OH)2+ cations migrate toward the negative pole, while the CrO42‒, Cr2O72‒ anions
of Cr(VI) migrates toward the positive pole. If laminar flow is maintained in the capillary
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towards the negative pole of CE instrument, the analytical signal of the two forms can be
registered in the same chromatogram.
The CE and ICP-MS instruments had been coupled so that the CE quartz capillary were plugged
into the high efficiency nebulizer (HEN) of ICP-MS, while the negative pole of electrophoretic
system was connected to the nebulizer. The HEN nebulizer sucking effect had created a 1
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l/min laminar flow that made it possible for Cr (III) and Cr (VI) species to enter the ICP-MS
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after each other consequently.
1.7. Separation of Cr (III) and Cr (VI) using electrothermal vaporization
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In an other group of on-line speciation techniques’ separation is carried out based on the
difference in thermal properties, boiling points and volatility of species, instead of
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chromatographic techniques. The species gradually leave the heated sample compartment as
temperature rises into the element-selective detector.
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Cr (III) compounds reacts with certain acetylacetonates to give volatile complex compounds
[12, 13]. These substances undergo sublimation even at 100 oC. 2-teonil-trifluoroacetonate
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(TTA) was chosen as complexing agent for chromium speciation purpose. This substance does
not react with Cr (VI) species.
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The atomizing unit of graphite furnace atomic absorption spectrometer was transformed into a
sample introduction system of electrothermal vaporization [14]. The sample droplet introduced
to the graphite tube of ETV system was subjected to drying, ashing, atomization similarly to
conventional heating program of GFAAS. The Cr (III)-teonyl trifluoroacetonate complex (CrTTA) is completely vaporized even in the ashing step at few hundred degree Celsius. The nonvolatile Cr (VI) species leaves the graphite tube only above 2000 oC in the atomization stage.
The ETV system has been developed so that the chromium species evaporated from the inner
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wall of graphite tube is flushed into a flame atomic absorption spectrometer (FAAS) with argon
stream.
2. Connection parts (interfaces) in on-line speciation analysis
A crucial point of atomic spectrometric techniques is the introduction of sample. The analytical
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performance of a speciation analytical technique highly depends on the rate and efficiency of
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sample introduction. In case of conventional pneumatic nebulizers of flame spectrometry, the
rate of sample introduction is 4-6 ml/min, the efficiency of sample introduction is 5 – 10 %.
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These values are 1-2 ml/min and 1-2 % for inductively coupled plasma atomic emission
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spectrometry (ICP-AES), respectively.
The appearance of hydraulic high pressure nebulization (HHPN) has significantly increased the
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efficiency of sample introduction of atomic spectrometric techniques, even 40-50 % efficiency
can be achieved for aqueous solutions, under 2 - 3 ml/min sample flow rate [15-17]. The
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increased liquid flow rate, however, is a disadvantage for ICP-AES and ICP-MS, since the
plasma may be extinguished. This adverse effect can be avoided by leading the wet aerosol
plasma.
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produced by HHPN nebulizer through a desolvation unit to minimize the solvent load of the
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The difference in flow rate needed for the separation system and for the nebulizer of elementselective detector might cause further difficulties in coupling of the two techniques. The online
coupling of capillary electrophoresis instrument and ICP spectrometer for instance is not
obvious, since the l/min range sample flow rate in CE capillary and the required few ml/min
flow rate of ICP spectrometers cannot be coupled directly. This difficulty can be overcome by
adding an auxiliary diluting liquid to the solution emerging from CE capillary.
The high efficiency of sample introduction by electrothermal vaporization is credited to the
formation of crystalline nanoparticles from the fumes of vaporized chromium species in the
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argon carrier stream. These particles can be carried to a relatively high distance without
considerable transport loss.
3. Element-selective detection units for on-line chromium speciation
Such atomic spectrometric methods were used for the development of on-line chromium
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speciation techniques, which provided continuous stationary sample introduction. The applied
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methods were flame atomic absorption spectrometry (FAAS), inductively coupled plasma
atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry
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(ICP-MS) and acetylene – nitrous oxide flame emission spectrometry (Ac/N2O-FES), whereas
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their analytical performances were compared to each other with respect to chromium speciation.
One possible way to increase the analytical performance of FAAS is the enrichment of the
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separated chromium species, or to increase the efficiency of sample introduction by using
HHPN nebulizer instead of conventional pneumatic nebulizer. We can similarly reach the same
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improvement in case of ICP-AES by enrichment of the separated chromium species.
Nevertheless the high organic material content of the eluent used for the chromium speciation
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requires the use of desolvation of the nebulized aerosol, before flushing it to the plasma.
The analytical performance of ICP-MS technique is highly influenced by the occurring isobar
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interferences in case of quadrupole analyzer. Chromium has 4 isotopes (the relative abundances
of isotopes is given in parenthesis): 50Cr (4.3%), 52Cr (83.5%), 53Cr (9.5%), 54Cr (2.4 %). The
most abundant isotope with atomic mass number 52 would be the best choice for the analysis
of samples containing chromium in ng/mL range. The 35Cl16OH and 40Ar12C molecular ions
causes a possible difficulty for the determination of 52Cr isotope. The mass number of 35Cl16OH
is 51, however, in case of sea water samples it might exert isobar interference due to the
extremely high chlorine concentration for 52Cr quadrupole MS determinations. The aerosol
entering the plasma has high organic material content, derived especially from methanol and
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TBA-salts used in chromium speciation, by this way the concentration of 40Ar12C molecular ion
is significantly increased. The resulted considerable isobar interferences make the quadrupole
MS analysis unfavorable using the 52 mass number chromium isotope. Furthermore chloride
atom containing species develops further isobar interferences for MS measurements of
chromium at 54 and 53 mass number isotopes, whereas the 40Ar13C imply further interference
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for the measurement at the latter Cr isotope. The most interference-free chromium isotope is
50
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Cr, which has only 4.3% natural abundance that reduces the limit of detection of ICP-MS with
more than one orders of magnitude.
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The flame emission spectrometry (FES) is not listed nowadays in the group of high sensitivity
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atomic spectrometric techniques despite of its historical importance in the development of
atomic spectrometry. Nevertheless flame emission spectrometry using high temperature (3000
C) acetylene – nitrous oxide flame is proved to be a powerful tool for determining chromium
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concentration for its speciation. The wavelength of the highest relative intensity emission line
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of chromium is 425.43 nm in case of acetylene – nitrous oxide flame [18], which is the first
line of the triplet appearing in the visible UV range (λ = 425.43 – 427.48 – 428.97 nm). This
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line is emitted as a result of 7S3 – z7P4o electron transition between the 0 – 23.499 kayser energy
levels. As a result the line has low, 2.91 eV excitation potential [19]. The lack of molecular
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bands from the corresponding low noise region of the spectrum of the flame is a further
advantage of the method.
4. Off-line chromium speciation methods
Nowadays one of the highest sensitivity atomic spectrometric technique is graphite
furnace atomic absorption spectrometry (GFAAS). The sample introduction in GFAAS is not
continuous, not stationary, but periodical. By this way the method cannot be used for the
development of on-line coupled techniques. The high sensitivity off-line element speciation
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techniques are relied on GFAAS. Several off-line chromium speciation techniques with high
analytical performance had been developed with GFAAS owing to its high sensitivity and low
sample demand, despite of the longer analysis time compared to on-line speciation techniques.
4.1. Separation/enrichment of Cr (VI) with continuous extraction
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The schematic arrangement of the developed semi-automatic system is shown in Figure 1. Its
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Fig 1.
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structure and working principle is based on a previously developed titration device [20, 21].
The sample to be extracted is placed in the sample compartment with the maximum volume of
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100 mL that is connected to mixing chamber through a 1 mm inner diameter glass capillary.
The mixing chamber contains the 2 mL immiscible organic solvent (like chloroform), with a
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density higher than water. The ion pair forming agent (like methyl trioctylammonium chloride)
is dissolved in the organic phase. The magnetic stirrer rod rotating at a high rate disperses the
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organic solution into small droplets to increase the efficiency of liquid-liquid extraction.
The mixing chamber and the connected buffer vessel is filled up with ion-exchanged water until
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the end of metal capillary is immersed in the water. The buffer vessel is sealed with a glass plug
containing a stainless steel capillary in its middle. This metal capillary can be connected to the
nebulizer of FAAS instrument, or to a peristaltic pump. Both of the above mentioned methods
assure the chromium species containing sample solution to pass-through the mixing chamber,
containing the organic solvent where the extraction takes place. The concentration of chromium
(VI) enriched in the organic phase was determined by GFAAS. If the extraction device is
connected to an FAAS instrument, the concentration of chromium (VI) in aqueous phase can
be continuously monitored to follow the efficiency of the extraction.
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4.2. Enrichment of chromium species using single drop microextraction
The method of Single Drop Microextraction (SDME) is based on the formation of a single
hanging drop of organic solvent containing the complexing agent by a microsyringe in the
solution to be extracted [22]. The studied species form a complex that is extracted to the organic
phase by the mild agitation of sample solution, thus it is enriched in the drop.
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The microextraction can easily be coupled with GFAAS technique, since the enrichment
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requires only small amount (5 – 10 μL) of organic solvent, which can be drawn back to syringe
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after the extraction and injected directly to the graphite furnace
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4.2.1. Single drop microextraction enrichment of Cr (III) with oxine
Single drop microextraction were adapted first for the enrichment of Cr (III). Chloroform was
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used as organic phase, containing the dissolved complexing agent, oxine. The pH of sample
solution was adjusted to 8 with a buffer solution of ammonium hydroxide – ammonium chloride
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in the ratio of 1:2. A drop of chloroform containing 0.1 mol/L oxine was formed in the cell
using a microsyringe. The Cr (III) content of the sample solution was enriched in the drop in
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form of chromium (III)-oxine complex. After the extraction the drop was drawn back to the
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syringe and it was injected directly to the graphite furnace of GFAAS.
4.2.2. SDME enrichment of Cr (VI) with tridecylmethylammonium chloride
complexing agent
Tridecylmethylammonium chloride, an ion-pair complexing agent with good solubility in
chloroform was chosen for the SDME enrichment of Cr (VI). This complexing agent forms a
stabile complex with Cr (VI) in the pH range of 2-5. The rate of complex formation is fast and
the formed complex dissolves well in chloroform, by this way this reaction can be the basis of
an SDME chromium speciation technique.
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4.3. Enrichment of Cr (VI) with modified SDME method [23]
The presented SDME method has a disadvantage beside its favorable analytical performance
that is the hanging drop can easily be carried away by the sample flow passing through the
enrichment cell. The volume of the formed drop at the tip of the microsyringe needle is less
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than 3 L if chloroform is used as organic solvent. The lossless handling and precise injection
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of such a small sample volume might be a potential source of experimental error. Furthermore
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the optimal sample volume for GFAAS measurement is 20 – 30 L. The limit of detection for
GFAAS measurement could be improved by one orders of magnitude if the volume of the
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enriching drop would be in this volume range. The original hanging drop microextraction
technique had been modified according to the above guidelines. In the modified system the drop
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of organic solvent is not hanging from the tip of needle, but it is sitting in a designed cavity at
the bottom of the extraction cell [23]. By this way the breaking down and the consequent loss
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GFAAS measurements.
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of the drop can be avoided, and the volume of the drop (20 – 30 μL) is more favorable for the
4.4. Adaptation of chromium speciation methods for GFAAS determination
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Most frequently the following complex forming agent had been used for spectrophotometric
chromium speciation methods: diphenylcarbazide [24], crystal violet [25], pentamethylene bistriphenylphosphonium [26], and other dyes [27]. Our goal was to find new complexing agents
that selectively form stable complexes with one of the chromium species, which can be
dissolved in organic solvents. The spectrophotometric determination of chromium was replaced
by a high sensitivity element-selective detector, graphite furnace atomic absorption
spectrometry (GFAAS).
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4.4.1. Liquid-liquid extraction of Cr (VI) – DIC complex [28]
The dimethylindocarbocyanide (DIC) dye and its chromium (VI) complex is stable even at
room temperature, unlike other chromium complexes [27]. The molar extinction coefficient of
the complex is relatively high, still it is not relevant for GFAAS measurement, since the analysis
is based on the light absorption of free chromium atoms liberated from the complex in the
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graphite furnace. Toluene was chosen as organic phase for the extraction.
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Fig 2.
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4.4.2. Liquid-liquid extraction of Cr (VI) using MPVTI complexing agent [29]
A new organic compound, 2-[2-(4-methoxy-phenylamino)-vinyl]-1,3,3-trimethyl-3H-indolium
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chloride (MPVTI) was proposed as complex forming agent for enrichment and
spectrophotometric determination of Cr (VI) by Andruch et al. [30]. The structure of the reagent
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is shown in Figure 3.
Fig 3.
The reagent forms a stable complex with Cr (VI) in acidic media in the presence of chloride
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ion. The complex was extracted to toluene.
4.4.3. Liquid-liquid extraction of Cr (VI) by the formation of diperoxo-chromium
complex
The Cr(VI) species is transformed into a blue color diperoxo-chromium complex with hydrogen
peroxide under appropriate conditions (Figure 4.). The complex can be extracted to organic
phase (in our case to ethyl acetate)
Fig 4.
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Diperoxo-chromium is an unstable substance at room temperature, can easily be reduced to Cr
(III). Thus the extraction was carried out in a mixture of water-ethyl acetate cooled below 10
C. The ratio of water – ethyl acetate was 5:1 (10 mL aqueous sample – 2 mL ethyl acetate) to
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achieve a 5-fold dilution of Cr (VI) in the organic phase. It could be concluded from method
optimization process that highest efficiency extraction can be achieved when the pH of sample
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solution is set to 1,7± 0.1 with sulphuric acid and the concentration of hydrogen peroxide is
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0.02 mol/L. Further extraction steps can be performed for the further enrichment of Cr (VI).
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5. Comparison of analytical performances of chromium speciation methods
The limit of detection values of the developed on-line and off-line chromium speciation
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methods are shown in Table 1 that is compared to the chromium concentration of some natural
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samples together with the range of measurement of the methods in Figure 5. The meaning of
symbols and abbreviations are given in Figure and Table legends.
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On-line separation
Chromium
form
Cr(VI)
Cr(III)
Cr(VI)
Cr(VI)
Cr(III)
Cr(VI)
Cr(VI)
Cr(III)
Cr(VI)
Cr(VI)
Cr(VI)
Cr(VI)
Cr(VI)
Cr(III)
Cr(VI)
Cr(VI)
Cr(VI)
Cr(VI)
On-line
preconcentration
off-line separation
off-line
preconcentration
Separator
unit
C-18 TBA-salt
C-18 TBA-salt
C-18 TBA-salt
C-18 TBA-salt
C-18 TBA-salt
CE
PEEK loop APDC
C-18 KH-phtalate
PEEK loop APDC
PEEK loop APDC
C-18 TBA-salt
C-18 TBA-salt
C-18 TBA-salt
C-18 KH-phtalate
LLE DIC
LLE MPVTI
Modified SPME
Continuous LLE
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Method of
speciation
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Table 1. Limit of detections of the developed chromium speciation techniques for the various
on-line and off-line separation/enrichment methods using different sample introduction and
detection units
Sample
introduction
PN
HHPN
HHPN
US - DES
US - DES
PN-HEN
PN
HHPN
HHPN
HHPN
US - DES
US - DES
HHPN
HHPN
Detection
FAAS
FAAS
FAAS
ICP-AES
ICP-AES
ICP-MS
FAAS
FAAS
FAAS
FAAS
ICP-AES
ICP-MS
FES (Ac/N2O)
FES (Ac/N2O)
GFAAS
GFAAS
GFAAS
GFAAS
LOD (3σ)
ng/mL
80
30
20
3.7
4.6
24
2
0.92
0.54
0.073
0.36
0.12
0.020
0.025
0.25
0.15
0.042
0.010
Ref.
[31]
[5]
[5]
[32]
[32]
[10, 11]
[8, 33]
[34]
[34]
[34]
[32]
[32]
[35-37]
[35-37]
[28]
[29]
[23]
[21]
C-18 = C-18 reversed-phase high pressure chromatographic column, TBA-salt = tetrabuthyl ammonium salt, CE
= capillary electrophoresis, PEEK loop = poly-ether-ether-ketone loop, APDC = ammonium
pyrrolidindithyocarbamate, LLE = liquid-liquid extraction, DIC = Dimethyl indocarbocyanide, MPVTI = 2-[2-(4methoxy phenilammino)-vynil]-1,3,3-trimethyl-3H-indolium chloride, SPME = single drop microextraction,
PN= pneumatic nebulization, US – DES = ultrasonic nebulization and desolvation, HHPN = hydraulic high
pressure nebulization ,
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FAAS = flame atomic absorption spectrometry, ICP-AES = inductively coupled plasma atomic emission
spectrometry, ICP-MS = inductively coupled plasma mass spectrometry, FES (Ac/N 2O) = nitrous oxide
/acethylene flame emission spectrometry, GFAAS = graphite furnace atomic absorption spectrometry
Fig 5.
One of our developed method was compared with other studies (Table 2.) and tested with real
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samples (Table 3.).
species
RSD %
LOD µg·L-1
enrichment
factor
Cr(III)
0,1
0.15
1
Cr(VI)
2
0.003
Cr(III)
Cr(VI)
2.3
4
0.05
0.3
Cr(III)
1.2
0.01
Cr(III)
1
Cr(III)
Cr(VI)
5.6
2.1
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our and other studies
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Table 2. The comparison of the RSD%-s, limit of detections and enrichment factors between O
detection
Reference
tea, water
fluorimetrya
[38]
500
water
SPE-FAASb
[39]
48
30
water
FI-SPE-FAAS
[40]
200
water, soil
SPE-FAAS
[41]
0.32
25
water, soil
LLE-FAAS
[42]
0.025
0.020
86
50
water
FES (Ac/N2O)
This work
[35-37]
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sample
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MD-SPE: magnetic dispersive solid phase extraction
SPE-FAAS: solid phase extraction - flame atomic absorption spectrometry
FI-SPE-FAAS: flow injection-solid phase extraction- flame atomic absorption spectrometry
LLE-FAAS: liquid-liquid extraction-flame atomic absorption spectrometry
FES (Ac/N2O): flame emission spectrometry use in acetylene/dinitrogen oxide flame
a
The fluorimetry method can be used for As(III) determination, too [43]
b
The SPE sample preparation of this method can be used for As(III) determination, too [44]
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Table 3. Concentrations of the chromium species in various samples (n = 3a)
Concentration (μg L-1)
Cr(III)
0.44 ± 0.02b
Cr(VI)
Total Cr
0.19 ± 0.01
Tap water (Karcag)
0.15 ± 0.01
̶
0.63 ± 0.03
0.15 ± 0.01
Well water (Karcag)
1.5 ± 0.1
0.50 ± 0.02
2.0 ± 0.12
Sea water (Varna)
0.47 ± 0.02
0.21 ± 0.02
0.68 ± 0.04
Cigarette ash 1
Cigarette ash 2
Concentration (μg g-1)
2.09 ± 0.22
0.08 ± 0.01
2.17 ± 0.23
1.21 ± 0.18
0.07 ± 0.01
1.28 ± 0.19
Sample
c
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Tap water (Debrecen)
a
Number of replicate analyses.
At 95% confidence level (mean ± t*s /√3), s: standard deviation for the measurement,
t: Student's t-value
c
Below the detection limit. (LoD = 3/S), σ = standard deviation of blank value (n=10),
S = mean of slopes of the calibration curves
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b
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6. Conclusion
The advantage of on-line speciation techniques is their relatively short analysis time. The
result of the analysis can be achieved rapidly after the separation and enrichment of the element
species. The analytical performances of on-line speciation techniques can be improved at three
sections of the coupled separation/detection system. The first section is the possible enrichment
during the separation, where the selected element form is preconcentrated from a sample
volume larger than the conventional volumes used for separation, as a result more analyte enter
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the detector in a given volume of solution depending from the degree of enrichment, increasing
the signal-to-noise ratio.
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Similar improvement can be achieved with the increase of efficiency of sample
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introduction. When the conventional pneumatic nebulization is replaced by ultrasonic
nebulization or hydraulic high pressure nebulization, the efficiency of nebulization of aqueous
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solutions is increased to 15-20 % and 35-40 % from 1-10 %, respectively
The third section is the element selective detector, the analytical performance can be
further improved with better sensitivity detectors.
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The improvement in limit of detection of the demonstrated chromium speciation
techniques in Table 1 confirms the positive effect of change of the three sections of on-line
systems. The analytical performance of separation techniques without enrichment can be
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improved using more effective sample introduction techniques and more sensitive detectors.
According to the data of Table 1, 20-fold improvement in the limit of detection can be achieved
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when ultrasonic (US) or hydraulic high pressure nebulization (HHPN) is applied instead of
pneumatic nebulization (PN) and when flame atomic absorption spectrometry (FAAS) is
system..
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replaced by inductively coupled plasma atomic emission spectrometry (ICP-MS) as detection
The CE – ICP-MS has a special place among other sensitive speciation techniques.
Inductively coupled plasma mass spectrometer is one of the highest sensitivity detection
system. The electroosmotic flow (EOF) obtained during capillary electrophoretic separation
(CE) results only in nL/min range sample stream, which results in a moderate concentration
sensitivity, however, the mass sensitivity of the coupled system is quite high.
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The limit of detection of on-line techniques using FAAS detection system is significantly
improved when the chromium species are enriched, not only separated, according to Table 1.
0.1 ng/mL limit of detection can be achieved when ultrasonic nebulization is combined with
desolvation (US-DES) and ICP-MS detection unit is used. This coupled technique still has some
potential for further improvements. When hydraulic high pressure nebulization and desolvation
(HHPN-DES) and the more expensive double focusing mass spectrometry or time of flight
(TOF) mass spectrometry would have been used, the more sensitive 52Cr isotope could have
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been measured instead of low natural occurrence 50Cr isotope. These changes would result in
better limit of detection with approximately two orders of magnitude.
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Two limit of detection values are represented in Table 1 for enrichment of chromium (VI)
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with ammonium pyrrolidine dithyocarbamate in a PEEK loop. The sample volume used for
enrichment was 5 mL in one case, and this data was 50 mL in other case. The limit of detection
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of Cr (VI) improved from 0.54 ng/mL to 0.073 ng/mL. On the other hand, the analysis time of
on-line techniques increases with increasing sample volumes.
An unexpected discovery was made in chromium speciation. The best limit of detection
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for chromium (20 pg/mL for Cr(VI)), 25 pg/ml for Cr(III)) can be achieved by acetylene –
nitrous-oxide flame emission at the 425.4 nm line of chromium. This is a remarkable result,
since flame emission spectrometry is the cheapest technique among the introduced detection
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methods and it is more sensitive to chromium than the more expensive interference-sensitive
ICP-MS.
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The time and work demand of off-line techniques is larger, however, they have very good
limit of detections in chromium speciation, since graphite furnace atomic absorption
spectrometry (GFAAS) is similarly powerful detection unit like ICP-MS. Furthermore, GFAAS
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instruments are significantly cheaper and it has less sample demand (20 – 30 μL). The
adaptation of earlier liquid–liquid extraction techniques using spectrophotometric detection to
GFAAS detector results in high sensitivity speciation techniques. In case of enrichment the
limit of detection of off-line methods might even overcome the on-line techniques. The last line
of Table 1. demonstrates our best limit of detection for Cr (VI) (10 pg/mL) using GFAAS after
50-fold enrichment.
The achievement of best possible limit of detection has significant practical importance,
since the chromium level of natural environmental samples are rather low. In order to perform
reliable measurements in this concentration range, the limit of detection of the speciation
technique should be under the chromium level in natural samples and the measurement range
should fit this concentration range.
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Figure one demonstrates how the limit of detection and range of determination of each
developed techniques related to the concentration of some important natural samples.
According to Figure 5 two out of our developed techniques are capable for the measurement of
each type of samples, meeting the above mentioned criteria. One of them is the on-line
enrichment technique using hydraulic high pressure nebulization and acetylene –nitrous oxide
flame emission technique which was excellent for the chromium speciation of drinking water
samples. The other one is the off-line technique using graphite furnace atomic absorption
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spectrometry with similar outstanding analytical performance after an appropriate enrichment
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of sample solution.
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Acknowledgments
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The work is supported by the EFOP-3.6.3-VEKOP-16-2017-00008 project. The project is cofinanced by the European Union and the European Social Fund. Financial support from the
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Hungarian Research Foundations OTKA Grant T4508, 020235/1996, D29100; MKM FKFP
1045/1997 and 2249/1998; OTKA-POSTDOC 1998; DAAD 1992 are gratefully
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acknowledged.
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Figure caption
Figure 1. The continuous, semi-automatic liquid-liquid extraction device. 1 buffer vessel, 2
aqueous phase, 3 organic phase, 4 magnetic stirrer rod, 5 mixing chamber, 6 sample
compartment, 7 sample solution, 8 glass capillary, 9 teflon capillary to the nebulizer of flame
atomic absorption spectrometer (on-line operation method) OR to peristaltic pump and 10
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collection vessel (off-line operation method)
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Figure 2. Chemical structure of dimethylindocarbocyanide (DIC)
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Figure 3. Chemical structure of 2-[2-(4-methoxy-phenylamino)-vinyl]-1,3,3-trimethyl-3Hindolium chloride (MPVTI)
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Figure 4. The chemical structure of diperoxo-chromium (chromium (VI) oxide-peroxide)
Figure 5. Comparison of analytical performances of the developed on-line and off-line
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chromium speciation techniques with respect to level of chromium (VI) in some natural samples
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Table 1. Limit of detections of the developed chromium speciation techniques for the various
detection units
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on-line and off-line separation/enrichment methods using different sample introduction and
Table 2. The comparison of the RSD%-s, limit of detections and enrichment factors between our
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and other studies
Table 3. Concentrations of the chromium species in various samples (n = 3a)
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Figure 1. The continuous, semi-automatic liquid-liquid extraction device. 1 buffer vessel, 2
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aqueous phase, 3 organic phase, 4 magnetic stirrer rod, 5 mixing chamber, 6 sample
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compartment, 7 sample solution, 8 glass capillary, 9 teflon capillary to the nebulizer of flame
atomic absorption spectrometer (on-line operation method) OR to peristaltic pump and 10
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collection vessel (off-line operation method)
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Figure 2. Chemical structure of dimethylindocarbocyanide (DIC)
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indolium chloride (MPVTI)
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Figure 3. Chemical structure of 2-[2-(4-methoxy-phenylamino)-vinyl]-1,3,3-trimethyl-3H-
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Figure 4. The chemical structure of diperoxo-chromium (chromium (VI) oxide-peroxide)
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Figure 5. Comparison of analytical performances of the developed on-line and off-line
chromium speciation techniques with respect to level of chromium (VI) in some natural
samples = limit of detection,
= range of measurement
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Highlights
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
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20 various chromium speciation techniques had been developed with atomic spectrometric
detection
the developed methods are cheap and they have low labor and time demand
the achieved lowest limit of detection for Cr (VI) is 20 pg/mL for on-line and 10 pg/mL for
off-line techniques
the methods are capable for the chromium speciation analysis of natural samples
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