A high-throughput and high peak capacity narrow-bore parallel segmented flow column strategy for the liquid chromatography-tandem mass spectrometry analysis of organic contaminants in water

Arianne Soliven ab, Lucia Pareja c, R. Andrew Shalliker d, Horacio Heinzen ac and Andrés Pérez-Parada *e
aGrupo de Análisis de Contaminantes Traza, Cátedra de Farmacognosia y Productos Naturales, Facultad de Química, Universidad de la República, General Flores 2124, 11800 Montevideo, Uruguay
bAdvanced Materials Technology, Inc., 3521 Silverside Road, Suite 1-K, Quillen Building, Wilmington, DE 19810, USA
cDepartamento de Química del Litoral, CENUR Litoral Norte, Universidad de la República, Ruta 3 Km 363, Paysandú, Uruguay
dAustralian Centre for Research on Separation Science (ACROSS), School of Science and Health, Western Sydney University, Parramatta, NSW 2150, Australia
eDepartamento de Desarrollo Tecnológico – DDT, Centro Universitario Regional del Este (CURE), Universidad de la República, Ruta 9 y Ruta 15, 27000, Rocha, Uruguay. E-mail: aperez@cure.edu.uy

Received 2nd November 2019 , Accepted 21st December 2019

First published on 23rd December 2019

This study highlights the development of a high peak capacity, high-throughput (HTP) approach for a target list of 62 organic contaminants in an environmental river water matrix on a HPLC conventional 400 bar system. Key separation metrics were evaluated: (a) peak capacity, (b) total analysis time and (c) mobile phase consumption. An average peak width of 0.10 min with a total analysis time (inclusive of the column wash and re-equilibration) of 10 minutes revealed the increased productivity and performance of the parallel segmented flow (PSF) column technology HPLC-MS strategy, comparable to UHPLC peak widths and analysis times, achieved at a significantly lower backpressure (<400 bar). The operation of the PSF in a narrow-bore scale format (internal diameter of 2.1 mm) resulted in a conservative HPLC scale total mobile phase consumption of 15 mL for the column separation; only 5.5 mL of this volume was exposed to the ion source per injection. Mobile phase consumption was higher compared to that of UHPLC, but on the other hand it achieved higher peak capacity. Three representative compounds (atrazine, diclofenac and fluazuron) with differing retention and ionisation properties were studied in detail in terms of detection sensitivity. The majority of the ion ratios for the standards in the river water matrix were within ±30% of the average ion ratios. The largest ion suppression occurred for atrazine (−25% matrix effect), a pesticide notorious for poor ionization and poor peak shape issues. The lowest response transitions at 1 μg L−1 for the extracted ions in the river water matrix of atrazine, diclofenac and fluazuron had signal to noise ratios ≥3, with the exception of diclofenac where 5 μg L−1 was the lowest calibration level. The peak area's calibration curve slope and standard deviation in the detector's response determination of limit of quantification (LOQ) were between 5.2 and 30.4 μg L−1.

1. Introduction

High-throughput HPLC analyses achieve shorter run-times via increased velocities, which in turn are not ideal for coupling with MS detection that operates at lower volumetric flowrates.1 Microflow and nanoflow scale liquid chromatography systems coupled to mass spectrometry detection techniques have been presented as viable approaches for the reduced flow rate interface between LC and MS for the analysis of organic pollutants in complex samples in environmental and food matrices.2–5 However, these techniques rely on costly analytical instrumentation for pumping, injection and ionization processes. On the other hand, 400 bar HPLC is one of the most highly disseminated techniques and probably default instrumentation at any scientific laboratory due to its robustness and relatively lower maintenance in comparison with UHPLC.6 An alternative to reduce the volumetric flow while operating on the robust 400 bar HPLC system is to employ parallel segmented flow column technology.1

The parallel segmented flow (PSF) column technology utilises a special column end-fitting that has been designed to remove the wall effect and radial flow heterogeneities that are associated with imperfect packing and viscous frictional heating effects.7–9 The use of PSF column outlet is also an alternative interfacing strategy for managing the fluid between the HPLC and MS detector for high-throughput column separations coupled with relatively lower volumetric flowrates to the inlet of the electrospray ionization (ESI) source.1

Since the first introduction of the PSF column in 2012, only one study has demonstrated its potential application in trace analyses:10 this preliminary study utilized analytical scale (i.e., 4.6 mm i.d.) PSF column technology operating under isocratic conditions, representative of the isocratic holds often employed under gradient elution conditions for increased chromatographic resolution between co-eluting species. Additionally, for the first time, the isocratic conditions provided insight on the MS detection-based results where the changing mobile phase composition did not influence the ionisation process. The PSF column technology outperformed conventional column technology when the average particle diameters (dp) were matched (3 μm). The peaks eluting from the PSF column were significantly reduced in width (up to 70%)10 and because the mobile phase flow stream was radially split, the flow through the PSF column could be increased such that the volumetric mobile phase flowrate directed to the MS detector was exactly the same as that when a conventional column was employed. In effect, the mobile phase flowrate through the PSF column was increased 2.5-fold compared to the conventional column, increasing the analytical through-put with limit of quantification (LOQ) values comparable to the conventional column.

In this study we have continued to focus on exploiting the advantages of PSF column technology to achieve high-throughput and high peak capacity assays with operation at less than 400 bar. Further, in this study we have utilised narrow-bore columns in order to reduce the HPLC mobile phase consumption and subsequent disposal. The reduction of the HPLC column formats from analytical to narrow-bore formats (4.6 to 2.1 mm i.d.) significantly decreases the solvent/reagent consumption and waste generation (80% decrease)11 for a more environment-friendly and reduced cost procedure.12

The applicability of the PSF technology in trace analyses is demonstrated here via a complex separation targeted towards contaminants in an environmental river water matrix utilizing conventional HPLC-ESI-MS/MS equipment. A separation of 62 compounds that varied in hydrophobic and ionization properties, representative of different classes of chemicals used as pesticides, pharmaceuticals, and veterinary drugs, was performed. Potential markers may indicate agricultural run-off, industrial effluent/waste and/or sewerage plant contamination monitored in river water. Key separation metrics were evaluated: (a) peak capacity, (b) total analysis time and (c) mobile phase consumption.

2. Experimental

2.1. Chemicals

All mobile phases were prepared from HPLC-grade acetonitrile (ACN) supplied by J.T. Baker (Darmstadt, Germany). Milli-Q water (18.2 MΩ cm) was prepared via a Barnstead Easy Pure RoDi water system from Thermo Fisher Scientific (Waltham, MA, USA). Formic acid was purchased from Merck KGaA (Darmstadt, Germany). The full list of 62 contaminant standards including pesticides, anti-inflammatories and veterinary drugs is shown in Table 1. Analytical standards in a purity range of 96–99.9% were supplied by HPC Standards GmbH (Berlin, Germany) and Dr. Ehrenstorfer (Augsburg, Germany). The sample preparation solvents were prepared from HPLC grade methanol (MeOH) supplied by J. T. Baker (Darmstadt, Germany). Acetic acid was purchased from Dorwill (Buenos Aires, Argentina). Magnesium sulfate (MgSO4) and primary secondary amine (PSA) sorbent were purchased from Scharlab S.L. (Barcelona, Spain). Sodium acetate 3-hydrate was purchased from J.T. Baker (PA, USA). All materials were used as received.
Table 1 List of contaminants and their retention times (RT), and peak widths using a PSF narrow-bore (100 × 2.1 mm, 5 μm dp) column at the 10 μg L−1 level in the river water matrix
Name RT (min) Peak width (min) Name RT (min) Peak width (min)
Omethoate 0.42 0.10 Azinphos methyl 4.38 0.10
Carbendazim 0.92 0.08 Spinosad A 4.71 0.09
Levimisole 0.98 0.08 Methoxyfenozide 4.81 0.10
Azaterol 1.55 0.20 Isoprothiolane 4.82 0.10
Imazapyr 1.74 0.10 Tetraconazole 4.83 0.10
Thiamethoxam 1.83 0.10 Diclofenac 4.84 0.10
Danofloxacin 2.12 0.10 Alachlor 4.87 0.20
Clothianidin 2.13 0.10 Pirimifos methyl 4.88 0.12
Enrofloxacin 2.17 0.08 Metconazole 4.92 0.10
Clembuterol 2.19 0.10 Malathion 4.92 0.10
Imidacloprid 2.23 0.09 Propiconazole 4.98 0.15
Dimethoate 2.27 0.09 Triclabendazole 5.05 0.10
Acetamiprid 2.48 0.09 Emamectin Benzoate 5.23 0.10
Imazethapyr 2.67 0.10 Kresoxim methyl 5.26 0.10
Thiacloprid 2.85 0.08 Diazinon 5.29 0.10
Ametryn 2.87 0.08 Difenoconazole 5.34 0.12
Mebendazole 3.10 0.08 Pyraclostrobin 5.53 0.09
Propoxur 3.23 0.10 Coumaphos 5.56 0.10
Malaoxon 3.37 0.09 Chlorpyrifos 5.66 0.10
Metsulfuron methyl 3.41 0.08 Haloxifop methyl 5.78 0.10
Atrazine 3.42 0.10 Trifloxystrobin 5.92 0.10
Carbaryl 3.54 0.08 Eprinomectin 6.15 0.10
Flutriafol 3.68 0.09 Hexythiazox 6.26 0.10
Flunixin 3.69 0.10 Fluazuron 6.27 0.10
Fenbendazole 3.69 0.10 Fluazifop 6.28 0.09
Clomazone 3.98 0.08 Pendimethalin 6.31 0.10
Chlorantraniliprole 4.06 0.10 Abamectin 6.46 0.10
Spiroxamine 4.09 0.10 Ethion 6.46 0.09
Propanil 4.12 0.10 Doramectin 6.81 0.09
Methiocarb 4.28 0.09 Moxidectin 7.34 0.10
Linuron d6 4.33 0.08 Monensin 7.43 0.14
Average peak width 0.10

2.2. Chromatography columns

Hypersil GOLD columns were supplied by Thermo Fisher Scientific (Runcorn, Cheshire, United Kingdom). Two columns were utilised, one in a conventional format and the other in the PSF format. Both were narrow-bore (100 × 2.1 mm) formats, and the conventional column was packed with particles with 3 μm dp, while the PSF column was packed with particles with a relatively larger dp of 5 μm. The selection of these two columns and velocities for the comparison of <400 bar HPLC conventional vs. PSF columns was based on a previous study that was strictly limited to the evaluation of a narrow-bore HPLC column's van Deemter characterisation (column packing, longitudinal diffusion and mass transfer kinetics) based on the second moment method of the peak.13 The conventional 3 μm dp comparison was operated at the highest linear velocity <400 bar that matched the plate height of the PSF column packed with particles with a larger dp of 5 μm.13 The evaluation of this study focused on key separation metrics: (a) peak capacity, (b) total analysis time, (c) mobile phase consumption – characterized by the complex separation of 62 compounds in a river water matrix.

2.3. Instrumentation

All chromatographic experiments were conducted using an Agilent 1200 LC system (Santa Clara, CA, USA) equipped with an Agilent 1200 auto-sampler and an Agilent 1200 Infinity binary pump with a 420 μL mixer. A Sciex 4000 QTRAP® MS/MS (Concord, Canada) was operated with an ESI Turbo V™ ion source, and all experiments were operated in ESI+ scheduled mode. The ESI source parameters were optimized for the largest conventional intensity response. The final ESI source settings were fixed for all analyses and optimized only for the conventional LC column and are as follows: ion source temperature 400 °C, ion spray voltage 4000 V, curtain gas: 20 psi, ion source gas 1: 50 psi, and ion source gas 2: 50 psi.

2.4. Standard and sample preparation

2.4.1. Standard preparation. The full list of 62 contaminant standards is listed in Table 1. Briefly, individual stock standard solutions of the target compounds were prepared, based on the solubility properties of each compound, in pure MeOH or ACN and stored at −18 °C. Each standard was weighed to approximately 20 mg and was dissolved in 10.00 mL of the appropriate solvent to obtain a 2000 mg L−1 stock solution. Working solutions (100 mg L−1 and 10 mg L−1) were prepared by appropriate dilution of the stock solutions in ACN and used for the construction of matrix matched calibration curves.
2.4.2. Water matrix sample preparation. A river water matrix was obtained from Rio Negro, Uruguay (−33.134443, −57.157523). According to traditional approaches, the river water matrix was subjected to a solid phase extraction (SPE) approach via Oasis™ HLB 200 cc. Two hundred millilitres of river water was passed through the SPE cartridge, followed by two 4 mL methanol portions, and concentrated to 1 mL.14

2.5. Chromatographic conditions

All injection volumes were 3 μL for the PSF column. All separations were performed under gradient conditions. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. A linear gradient was used starting at 0 min with a change from 5% to 100% of mobile phase B in 9 min. At the end of the gradient the column was held at 100% B for 0.5 minutes and then returned to its initial conditions (5% B) in 0.1 min. The system was re-equilibrated in the initial mobile phase for 0.4 min prior to the next injection. The total mobile phase consumption of 15 mL for the column separation and only 5.5 mL was exposed to the ion source per injection. The connection tubing from the column outlet of the conventional column and the central port of the PSF column connected to the inlet of the MS ESI ion source was minimized using 10.5 cm of 0.0025′′ i.d. inch peek tubing (total volume of 0.3 μL). The volumetric flowrate through the PSF column was 1.5 mL min−1 with a PSF split ratio of 37% allowing just 0.555 mL min−1 from the radial central exit port, which was directed to the MS detector. The back-pressure control (via peek tubing) and PSF segmentation ratios were within the optimum operational performance region, systematically studied to be within 20–45% eluting from the central port.11 Subsequently, the PSF outlet split ratio affects the amount of the injection volume directed to the MS ion source inlet; 37% of the 3 μL auto-sampler injection volume was directed to the inlet of the ion source (approximately 1 μL).

The conventional column conditions had an injection of 3 μL and operated at a velocity of 300 μL min−1 – a crossover point where the reduced velocity of 1.6 cm s−1 of both the conventional and PSF had comparable plate heights.13 Starting at 0 min with a change from 5% to 100% of channel B in 25 min. At the end of the gradient the column was held at 100% B for one minute and then returned to its initial channel B composition in 0.1 min. The system was re-equilibrated in the initial mobile phase for 4.0 min prior to the next injection.

2.6. MS parameters

All ESI-MS/MS parameters were fixed and operated in multiple reaction monitoring (MRM) mode. Scheduled analysis with a 1 second cycle time was selected based on the reproducibility of the signal (Fig. 1) and resulted in ≤0.1 min peak widths, comparable to UHPLC peak widths with lower backpressure (<400 bar). Optimized conditions and settings (selected MRM transitions, declustering potential (DP), collision energies (CE) and collision cell exit potential (CXP)) used in this study are listed in Table 2. Two or three transitions listed in Table 2 were used for identification purposes. The largest response was used as quantitative MRM transition.
image file: c9ay02370d-f1.tif
Fig. 1 Cycle time experiments – atrazine (m/z) 216 → 174 (0.8, 1.2, 0.6 and 1.0 s). Insets of the triplicate injections of 0.8 s and 1.0 s cycle times are shown.
Table 2 Optimized conditions and settings for MRM transitions of three selected compounds. Declustering potential (DP), collision energies (CE) and collision cell exit potential (CXP)
Analyte Precursor ion (m/z) Product ion (m/z) DP (V) CE (eV) CXP (V)
Atrazine 216.1 174.0 21 25 10
103.9 21 27 10
Diclofenac 296.0 215.0 30 27 10
214.0 30 45 10
250.0 30 19 10
Fluazuron 505.9 158.1 86 33 8
348.9 86 33 22
141.07 86 65 22

2.7. Data analysis

Analyst 1.5 software was used for data acquisition and analysis without further treatment (no smoothing applied). The slope and the standard deviation of the detection response (SDy) of the calibration curve linear fit (y = mx + c) were used to determine the LOQ (10 × SDy/slope)15via LINEST functions in Microsoft Office Excel 2011. This LOQ is used as the lowest calibration level (LCL).

The sample peak capacity was based on the separation window of the last retained solute subtracted by the first retained solute, divided by the average peak width.16

3. Results and discussion

This study evaluated the potential of a high-throughput and high peak capacity narrow-bore PSF column HPLC-MS strategy for trace environmental contaminants (pesticides, pharmaceuticals, and veterinary drugs) <400 bars, characterized by the peak capacity, total analysis time and mobile phase consumption. The separation profile of 62 contaminants in river water at 10 μg L−1 is shown in Fig. 2. The sample peak capacity was higher for the PSF column (88) than for the conventional column (72). The conventional column had a higher resolution advantage – separating four extra-peaks compared to the two of the PSF as indicated in Fig. 2. Upon closer inspection, specifically at the section of the separation profiles where the most co-elution occurred and only taking into account the peaks with a detection response >5000 counts per second (cps), the conventional column had separated five extra-peaks compared to the two of the PSF (ESI Fig. SF1). The PSF sample peak capacity of 88 was achieved within a total analysis time (inclusive of column wash and re-equilibration to initial conditions) in less than 10 minutes (Fig. 2, top inset) and an average peak width of 6 seconds (Table 1). This result is typical for UHPLC technology, but here the PSF column technology was HPLC vintage operating at less than 400 bar and packed with 5 μm particles.17
image file: c9ay02370d-f2.tif
Fig. 2 The separation profiles achieved using PSF (top inset) and conventional (bottom inset) columns. Stars indicated extra resolved peaks. The colour coded total ion chromatogram of the 10 μg L−1 concentration level of the complex mixture of contaminants in the river water matrix.

The retention time reproducibility of the separated standards in the river water matrix (≤0.6% RSD) and solvent (<0.2% RSD) were representative of the robustness of the narrow-bore PSF HTP HPLC separations (Table 3). The detection response peak area reproducibility was ≤11.1% RSD and peak height ≤ 14.8% RSD, which were within the acceptable criteria of <20%.18

Table 3 Narrow-bore HTP PSF detection and retention time reproducibility and average peak widths
Atrazine Diclofenac Fluazuron
Solvent River water Solvent River water Solvent River water
a Triplicate injections at 10 μg L−1.
% RSD peak areaa 4.99 6.79 11.12 9.89 4.12 8.13
% RSD peak heighta 3.80 8.62 9.10 7.58 12.34 14.81
Retention time (min) 3.38 3.41 4.79 4.81 6.24 6.27
% RSD retention time 0.00 0.60 0.18 0.32 0.12 0.23
Average peak width (min) in Solvent 0.10 0.10 0.08 0.09 0.09 0.08
LCL (10 × SDy/slope) peak area 5.17 16.64 27.34 15.36 30.44 18.56
LCL (10 × SDy/slope) peak height 17.40 22.91 26.64 43.08 35.66 25.63

The PSF HTP high peak capacity approach matrix matched calibration curves for the river water matrix in Fig. 3(a) showed linearity >0.99. The matrix effects for three representative analytes of different ionization properties and retention properties (atrazine, diclofenac and fluazuron) were studied in detail. Atrazine's peak area indicated ion suppression (−25%), while the other ions were within ±20% representative of minor matrix effects for both peak areas and heights.18 In contrast, the conventional matrix effects resulted with atrazine (peak area and height) within ±20% and diclofenac (−21%), and fluazuron (−28%) peak heights indicated medium ion suppression.

image file: c9ay02370d-f3.tif
Fig. 3 (a) PSF calibration and matrix effect results of standards in river water, peak areas (top row) and peak heights (bottom row) for atrazine, diclofenac and fluazuron (1, 5, 10, 50 and 100 μg L−1). (b) PSF ion ratios of the river water matrix based on ±30% average of 1, 5, 10, 50 and 100 μg L−1 atrazine, diclofenac and fluazuron; peak area (left-hand side) and peak height (right-hand side) for atrazine, diclofenac and fluazuron.

The majority of river water matrix ion ratios illustrated in Fig. 3(b) were within ±30% of the average ion ratios (ratio of the quantifier ion divided by the qualifier ion, and the average ratios for each analyte in the matrix at 1, 5, 10, 50 and 100 μg L−1 calibration points), with the exception of 1 μg L−1 fluazuron where both peak areas and peak height were just outside the limit. Atrazine at 1 μg L−1 peak area was just outside the lower limit while the peak height was within. The diclofenac 100 μg L−1 peak height ion ratio was also outside the lower limit while the peak area was within. The ion ratio limit was according to identification criteria used for pesticide residues in agricultural food and feed18 and outside the scope of this study to define a more appropriate set of acceptance criteria for routine use of organic contaminants in environmental matrices.

All signal to noise (S/N) transitions for atrazine, diclofenac and fluazuron at 1 μg L−1 were ≥3, while the only exception was for diclofenac where 5 μg L−1 was the lowest calibrant point to result in a S/N ≥ 3. The detection limits based on the calculation of the slope and the standard deviation of the detection response of the calibration curve LCL15 were within 5.2 to 30.4 μg L−1, respectively for atrazine, diclofenac and fluazuron peak areas (Table 3). The peak height LCL values were between 17.4 and 43.1 μg L−1.

The PSF HTP <400 bar methodology was developed for increased productivity and decreased peak width benefits while operating in a narrow-bore scale to conserve the mobile phase; this approach was not aimed to minimize the matrix effect, nor increase sensitivity. A matched MS volumetric flowrate study for trace analysis demonstrated comparable sensitivity and a 2.5 fold gain in productivity for the PSF column compared to a conventional column of the same column dimensions and particle diameter.10 In order to gain sensitivity an alternative column, the ‘curtain flow’ (CF) column should be utilized.7,10,19–21

The PSF approach separation metrics, peak capacity, total analysis time and mobile phase consumption, were compared to two existing UHPLC approaches. A method developed for the separation of 51 organic pollutants in river water had a total analysis time of 10 min,22 equal to the PSF HPLC approach. However, all 51 peaks eluted between 3.1 and 5.5 min for the UHPLC approach, despite having narrow peak widths of 0.1 min and not utilizing a larger separation window, resulted in a low peak capacity of 24. Compared to a ‘fast’ UHPLC approach for the separation of 23 contaminants in shellfish with a total analysis time of 15 min, the previously established study utilized a large proportion of their separation space, however suffered from broader peak shapes of up to 0.3 min and hence resulted in a relatively lower peak capacity of 32.23 The PSF approach was not only faster in terms of total analysis time, but also had sharper peak widths of 0.1 min. The PSF peak capacity of 88 was significantly higher than that of both previously established methods.

In terms of mobile phase consumption, both UHPLC methods consumed 60–80% lower volume compared to the narrow-bore PSF HPLC approach, which is expected for the different scales of chromatography when the linear velocities are translated into flowrates.24 HPLC compared to UHPLC consumes more mobile phase due to the larger column hold up volumes.24 Despite the lower mobile phase consumption advantages of UHPLC compared to HPLC, users must not under-estimate the maintenance cost of UHPLC compared to that of HPLC.6

The future perspectives of this PSF <400 bar column separation strategy for contaminant analyses are aimed at high productivity, high peak capacity LC separations coupled with a lower MS volumetric flowrate. PSF on a narrow-bore scale HPLC format enabled the conservative consumption of the mobile phase with this strategy limited to 15 mL per injection. The productivity gains of the PSF's high-throughput with a total analysis time of 10 min achieved an average peak width of 0.10 min, comparable to UHPLC peak widths without exceeding 400 bar.25 This study presents an attractive HPLC-MS hyphenation strategy that can maximise the velocity of the column separation and reduce the volumetric load to the MS inlet achieved by the simple replacement of the conventional column's end-fitting with the novel end-fitting of the PSF column.

4. Conclusion

This approach demonstrated the first high-throughput parallel segmented flow column technology strategy to exploit the robust separation of the HPLC system and deliver ≤0.1 min peak widths and the high peak capacity (88 in <10 minutes) approach for contaminant analyses <400 bar. Additionally, the narrow-bore PSF format enabled low HPLC scale mobile phase consumption of 15 mL per injection, and good compatibility with standard MS equipment with only 5.5 mL was interfaced from LC separation to ESI. Productivity is the main benefit of this strategy.

The largest matrix suppression result was obtained for atrazine (−25% matrix effect), a compound notorious for poor peak shape and ionization issues, with the HPLC-MS standard equipment employing the narrow-bore (2.1 mm i.d.) PSF column. The majority of the ion ratios were within the SANTE ± 30% of the average ratios. At 1 μg L−1 the lowest response transitions for the extracted ions in the river water matrix of atrazine, diclofenac and fluazuron had S/N ≥ 3, with the exception of diclofenac where 5 μg L−1 was the lowest level to achieve S/N ≥ 3.


PSFParallel segmented flow
LCLiquid chromatography
MSMass spectrometry
ESIElectro-spray ionization

Conflicts of interest

There are no conflicts to declare.


A. S. is a recipient of an ANII PD scholarship, and a researcher from CSIC and SNI, Uruguay. This research was supported by Comisión Sectorial de Investigación Científica, Universidad de la República.


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Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ay02370d

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