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Research Project
DEVELOPMENT OF AUTOMATIC MICROFLOW SYSTEMS FOR MONITORING MICROBIOGICAL, BIOCHEMICAL AND PHYSICO-CHEMICAL PARAMETERS IN BATHING WATERS
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Publications
Contributions to flow techniques and mass spectrometry in water analysis
Publication . Santos, Inês Carvalho dos; Rangel, António Osmaro Santos Silva; Mesquita, Raquel Beatriz Ribeiro de
In this thesis, the use of different flow systems was exploited along with the use of
different detection techniques for the development of simple, robust, and automated
analytical procedures. With the purpose to perform in-line sample handling and pretreatment
operations, different separation units were used. The main target for these
methods was waters samples. The first procedure was based on a sequential injection
analysis (SIA) system for carbon speciation (alkalinity, dissolved inorganic carbon (DIC),
dissolved organic carbon (DOC), and CO2) in bathing waters. The determination of
alkalinity was based on the reaction with acetic acid and corresponding color change of
bromcresol green (λ = 620 nm). The DIC, DOC, and CO2 determination was based in the
color change of bromothymol blue (BTB) indicator (λ = 660 nm) after the diffusion of the
gaseous CO2 through a hydrophobic membrane in a gas diffusion unit (GDU). For the DIC
determination, an in-line acidification prior to the GDU was needed. For DOC
determination, an in-line UV photo-oxidation of the sample was performed to convert all
carbon forms to CO2. A multi-reflective flow cell combined with a LED was used as
detection system enabling the minimization of the schlieren effect. The developed method
was successfully applied to inland bathing waters. The results were in agreement with the
reference procedure. The objective of the second work was the use of a SIA system to
determine iodide using potentiometric determination and iodate using a spectrophotometric
detection. Aiming to overcome the matrix interferences and cope with low analyte
concentration levels, the standard addition method was used. The incorporation of a mixing
chamber in a lateral port of the selection valve for a flow-batch approach enhanced the
mixture between sample, standards and reagent. The accuracy of the method was assessed
by comparing the obtained results with a reference procedure and using recovery tests. The
developed method was effectively applied to bathing waters and seaweed extracts. The
development of a multi-parametric system for cadmium and lead determination was the
objective of the third work of this thesis. A SI-LOV method with spectrophotometric
detection was used. Dithizone was chosen as the colour reagent as it forms a coloured
complex with both metals at different pH conditions. Cadmium determination was attained
at alkaline conditions while lead determination was attained at mid alkaline conditions.
The developed method was successfully applied to marine port waters and the results were
in agreement with the reference procedure. Subsequently, a solid phase spectrophotometry
method was developed for cadmium, zinc, and copper determination in freshwaters. The NTA resin was retained at the flow cell of the SI-LOV. After analyte retention, the
determination was based on the colorimetric reaction between the metal ions and dithizone.
Different retention and reaction pH were used to individually determine zinc and copper.
Cadmium concentration was calculated using a two equation system. The results obtained
were in agreement with the reference procedure. Afterwards, a flow injection analysis
system was developed for the determination of bromate in drinking waters. With the goal
of reaching low levels of bromate, a LWCC was coupled to the system. The
spectrophotometric determination was based on the oxidation of chlorpromazine by
bromate in an acidic medium, resulting in the formation of a coloured radical product. The
accuracy of the developed method was assessed with recovery studies. The objective of the
sixth work was to develop a cheaper and simpler method to monitor the biodegradation of
ciprofloxacin and ofloxacin. A monolithic column was coupled to a flow injection analysis
(FIA) system to separate both analytes. A spectrophotometer was used as detection system
(295 and 275 nm). The method was successfully applied to monitor the biodegradation of
both compounds by the strain Labrys portucalensis F11. Subsequently, a new MS-based
noncovalent binding determination method was developed. For that purpose, a continuous
stirred tank reactor (CSTR) was used as a flow injection device for exponential dilution of
an equimolar host-guest solution over time. By combining an exponential dilution model
with a previously established equimolar binding model, binding constants for host-guest
complexes were determined with a single injection. This methodology was applied for the
determination of binding constants between vancomycin and Ac-Lys(Ac)-Ala-Ala
tripeptide stereoisomers. The determination of anionic surfactants using an ESI-MS/MS
method was the objective of the eighth work. Different di-positive cationic reagents were
studied using Scan, SIM and MS/MS methods and the obtained detection limits (DLs)
were compared. The developed method was effectively applied for the determination of
perfluorooctanesulfonic acid, perfluorooctanoic acid, sodium dodecyl sulfate, stearic acid
and dodecylbenzenesulfonate in different water samples. The ninth work consisted in the
rapid identification of bacteria in potentially contaminated water using MALDI-TOF MS.
The possible application of the identified bacteria to bioremediation was evaluated by
determining their ability to degrade toluene and chloroform.
Determination of noncovalent binding using a continuous stirred tank reactor as a flow Injection device coupled to electrospray ionization mass spectrometry
Publication . Santos, Inês C.; Waybright, Veronica B.; Fan, Hui; Ramirez, Sabra; Mesquita, Raquel B. R.; Rangel, António O. S. S.; Fryčák, Petr; Schug, Kevin A.
Abstract. Described is a new method based on the concept of controlled band
dispersion, achieved by hyphenating flow injection analysis with ESI-MS for
noncovalent binding determinations. A continuous stirred tank reactor (CSTR) was
used as a FIA device for exponential dilution of an equimolar host-guest solution over
time. The data obtained was treated for the noncovalent binding determination using
an equimolar binding model. Dissociation constants between vancomycin and AcLys(Ac)-Ala-Ala-OH peptide stereoisomers were determined using both the positive
and negative ionization modes. The results obtained for Ac-L-Lys(Ac)-D-Ala-D-Ala (a
model for a Gram-positive bacterial cell wall) binding were in reasonable agreement
with literature values made by other mass spectrometry binding determination techniques. Also, the developed method allowed the determination of dissociation constants for vancomycin with AcL-Lys(Ac)-D-Ala-L-Ala, Ac-L-Lys(Ac)-L-Ala-D-Ala, and Ac-L-Lys(Ac)-L-Ala-L-Ala. Although some differences in
measured binding affinities were noted using different ionization modes, the results of each determination were
generally consistent. Differences are likely attributable to the influence of a pseudo-physiological ammonium
acetate buffer solution on the formation of positively- and negatively-charged ionic complexes.
Keywords: Binding constants, CSTR, Electrospray ionization mass spectrometry, Controlled band dispersion,
Vancomycin, Alanine stereoisomers
Iodine speciation in coastal and inland bathing waters and seaweeds extracts using a sequential injection standard addition flow-batch method
Publication . Santos, Inês C.; Mesquita, Raquel B. R.; Bordalo, Adriano A.; Rangel, António O. S. S.
The present work describes the development of a sequential injection standard addition method for iodine speciation in bathing waters and seaweeds extracts without prior sample treatment. Iodine speciation was obtained by assessing the iodide and iodate content, the two inorganic forms of iodine in waters. For the determination of iodide, an iodide ion selective electrode (ISE) was used. The indirect determination of iodate was based on the spectrophotometric determination of nitrite (Griess reaction). For the iodate measurement, a mixing chamber was employed (flow batch approach) to explore the inherent efficient mixing, essential for the indirect determination of iodate. The application of the standard addition method enabled detection limits of 0.14 µM for iodide and 0.02 µM for iodate, together with the direct introduction of the target water samples, coastal and inland bathing waters. The results obtained were in agreement with those obtained by ICP-MS and a colorimetric reference procedure. Recovery tests also confirmed the accuracy of the developed method which was effectively applied to bathing waters and seaweed extracts.
Use of solid phase extraction for the sequential injection determination of alkaline phosphatase activity in dynamic water systems
Publication . Santos, Inês C.; Mesquita, Raquel B.R.; Bordalo, Adriano A.; Rangel, António O.S.S.
In this work, a solid phase extraction sequential injection methodology for the determination of alkaline phosphatase activity in dynamic water systems was developed. The determination of the enzymatic activity was based on the spectrophotometric detection of a coloured product, p-nitrophenol, at 405 nm. The p-nitrophenol is the product of the catalytic decomposition of p-nitrophenyl phosphate, a noncoloured substrate. Considering the low levels expected in natural waters and exploiting the fact of alkaline phosphatase being a metalloprotein, the enzyme was pre-concentrated in-line using a NTA Superflow resin charged with Zn2þ ions. The developed sequential injection method enabled a quantification range of 0.044–0.441 unit mL 1 of enzyme activity with a detection limit of 0.0082 unit mL 1 enzyme activity (1.9 mmol L 1 of pNP) and a determination rate of 17 h 1. Recovery tests confirmed the accuracy of the developed sequential injection method and it was effectively applied to different natural waters and to plant root extracts.
Solid phase spectrometry in a micro SIA system for cadmium and lead determination
Publication . Santos, Inês C.; Mesquita, Raquel; Rangel, António O. S. S.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
Funding Award Number
SFRH/BD/76012/2011