One of the main difficulties that the scientific community faced in tackling issues related to emerging micropollutants is due to the fact that they are chemical compounds most often found in extremely low concentrations: only recently has it been possible to develop innovative analytical techniques able to detect them.
Substances currently under investigation in the PerFORM WATER 2030 project are numerous and they have been selected referring to the contamination of Metropolitan City of Milan through a two-step procedure:
a first phase of bibliographical research followed by a second phase of qualitative screening within CAP Group's wastewater treatment plants.
This preliminary activity allowed to identify some classes of micropollutants: 10 pharmaceuticals, 1 pharmaceutical transformation product and 1 industrial compound in wastewater;
5 fragrances and several trace elements detected in wastewater, suspended particulate matter and sludge samples.
The innovative analytical technology applied allowed to reach a high sensibility and specificity in measurments for each one of the considered compound.
For the detection of polar micropollutants, wastewater samples are pre-concentrated through a process called Solid Phase Extraction (SPE).
After a physical filtration, the samples (c. 500ml) are extracted through cartridges which contain a polymeric phase. Chemical interactions between the solid phase and aqueous matrix retain the organic compounds and eliminate the interferents. After washing, the elution is performed using a polar solvent that displaces the analytes from the solid phase into the final eluent. This is collected and evaporated under nitrogen stream until 1 ml final volume. This procedure concentrates the initial sample hundred of times.
After this pre-treatment phase, it is possible to perform the high performance liquid chromatography-mass spectrometry analytical method, which is commonly referred to by the acronym HPLC-MS/MS. To quantify molecules dissolved in a liquid phase, mass spectrometer coupled with liquid chromatography is the technique of choice.
HPLC-MS/MS is based on two key principles: the first is the separation of compounds solved in a complex mixture and it occurs during the chromatographic run. The analytical column, which contains a stationary phase, is flowed by the eluent in which the sample is mixed: chemical interactions between column solid phase and the mobile phase retain molecules in base on their chemical structure. Greater affinity means higher retention time (RT) in the column. The second principle is the selection of molecules on the basis of RT and molecular weight (m). The mass spectrometer doesn't measure the molecular mass but the ratio between mass and charge (m/z): the chemical ionization, achieved applying high voltage before the injection in the triple-quadrupole, produce a charged ion that can be detected in the analyzer. Once the parent ion is isolated in the first quadrupole, a collision gas breaks the parent and generates characteristic fragments that increase the specificity of detection system.
The final result returned by the instrument is a graph called chromatogram, in which measurements are returned as a function of time. The graph consists of a series of peaks that represent the elution of the individual analytes separated by the chromatographic process, as in the example shown here.
The addition of labelled standards before the filtration phase (internal standards distinguish from the native compounds because of different isotopic composition) allows to achieve a better quantification, correcting the matrix effect, which is relevant in wastewaters, and any errors that could have been done during the sample preparation or ionization.
Analyses of trace elements in wastewater are carried out for determination of total metals (i.e. including both dissolved and solid chemical species)
and of the dissolved fraction (i.e. after filtration with 0,45 μm pore size filters to remove the particulate fraction).
Analyses are carried out also on sewage sludge, after freeze-drying.
Samples are acidified by adding a proper mixture of HCl and HNO3 and microwave digested at high temperatures in closed vessels in order to dissolve elements present in particulate, colloidal and/or organic forms.
Determination of trace elements are carried out by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) and/or by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) with an octopole reaction and collision cell. In both methods liquid samples are nebulized and transported into the plasma, where atoms and ions reach excited states and then emit characteristic spectra of radiation. In ICP-OES, the radiation is detected and converted into an electrical signal that is used to identify the element that emitted that radiation. In ICP-MS, determination of trace elements in carried out by separation of ions according to mass/charge ratio. Signals can be converted into concentrations after construction of appropriate calibration curves.
Concentrations of mercury are determined by a direct mercury analyzer. After drying and decomposition at 750 °C, mercury vapors are released from samples and are adsorbed on a gold amalgam trap. With a subsequent temperature increase (at 900 °C) mercury vapors are released from the trap and concentrations are determination with an atomic absorption spectrophotometer at 253.65 nm wavelength. The analysis of Cr(VI) (Hexavalent chromium) is carried out on filtered wastewater samples (i.e. in the dissolved fraction). Cr(VI) in acid conditions reacts with diphenylcarbazide (C13H14N4O), forming a product with a characteristic purple-red color. Concentrations can be determined using a molecular absorption spectrophotometer at 540 nm wavelength.
To carry out the analysis of the fragrances on wastewater samples, it is necessary to first proceed to a preliminary filtration in order to separate the suspended solids from the water: the samples of water and sludge/suspended solids obtained are then analyzed separately with different methodologies.
A. Wastewater samples
Wastewater samples are filtered to separate suspended solids from water.
Aqueous matrix, after internal standard addition, is extracted with cartridges that can retain fragrances from water. After cartridges drying, compounds are recovered with appropriate solvents. Extracts are then concentrated using N2 flux and analyzed by GC-MS/MS. Compounds separation is achieved through the passage in a 60 m length column progressively heated.
Compounds reach the end of the column in different times and they are then analyzed by a mass spectrometer which can discriminate different molecules based on the mass/charge ratio of ions originated from their fragmentation. In water samples, galaxolide, galaxolidone and tonalide are the main compounds while celestolide and phantolide are detected only at trace levels
B. Sludge and suspended solids samples
Suspended solids separated from aqueous matrix are dehydrated in dryer until weight stabilization, while sludge samples are freeze-dried before analysis.
Fragrances are extracted from both matrix with an ultrasound bath adding internal standard directly to the extraction solvent.
After concentration, graphitized non-porous carbon is added to separate fragrances from impurities of both matrix.
Purified extracts are then filtered with syringe filters to separate carbon from samples, concentrated with N2 flux and analyzed in GC-Ion trap. The operating principle of this instrument is the same of GC-MS/MS but, in this case, column is 30m length.
Ion trap, used as a mass spectrometer, allows to retain and release different ions characteristic of the specific compound through the application of electromagnetic fields thus allowing molecule identification. In both matrix, galaxolide is the main compound.
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