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GREEN SAMPLE PREPARATION FOR LIQUID
CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS
OF ANIONIC AND CATIONIC ANALYTES
ABSTRACT
INTRODUCTION
EXPERIMENTAL
Reagents And Stock Solutions
Hydrogel Preparation
Weak Ion-Exchange SPE
RESULTS AND DISCUSSION
Environmental Considerations (Of SECS And SPE)
CONCLUSIONS
ACKNOWLEDGEMENT
Subscriber access provided by SUNY DOWNSTATE Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Technical Note GREEN SAMPLE PREPARATION FOR LIQUID CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS OF ANIONIC AND CATIONIC ANALYTES Alain Wuethrich, Paul Raymond Haddad, and Joselito P Quirino Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Apr 2015 Downloaded from http://pubs.acs.org on April 2, 2015 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. duration = 760 min organic solvent used = 108 mL duration < 45 min organic solvent used = 0 mL Solid-Phase Extraction steps 1. condition 2. equilibrate 3. load 4. wash 5. elute 6. dry 7. reconstitute Simultaneous Eletrophoretic Concentration and Separation HV steps 1. load 2. attach 3. apply voltage 4. transfer Page 1 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1
GREEN SAMPLE PREPARATION FOR LIQUID
CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS
OF ANIONIC AND CATIONIC ANALYTES
Alain Wuethrich, Paul R. Haddad, Joselito P. Quirino* Australian Centre for Research on Separation Science, School of Physical SciencesChemistry, University of Tasmania, Tasmania 7001, Australia *corresponding author: jquirino@utas.edu.au Page 2 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2
ABSTRACT
A sample preparation device for the simultaneous enrichment and separation of cationic and anionic analytes was designed and implemented in an 8-channel configuration. The device is based on the use of an electric field to transfer the analytes from a large volume of sample into small volumes of electrolyte that was suspended into two glass micropipettes using a conductive hydrogel. This simple, economical, fast, and green (no organic solvent required) sample preparation scheme was evaluated using cationic and anionic herbicides as test analytes in water. The analytical figures of merit and ecological aspects were evaluated against the state of the art sample preparation, solid-phase extraction. A drastic reduction in both sample preparation time (94% faster) and resources (99% less consumables used) was observed. Finally, the technique in combination with high performance liquid chromatography and capillary electrophoresis was applied to analysis of quaternary ammonium and phenoxypropionic acid herbicides in fortified river water as well as drinking water (at levels relevant to Australian guidelines). The presented sustainable sample preparation approach could easily be applied to other charged analytes or adopted by other laboratories. Page 3 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3
INTRODUCTION
A general concern of sample preparation is that it is often accompanied by a laborious and resource intensive workflow. Additionally, the use of toxic reagents and appreciable volumes of organic solvents further diminish the eco-friendliness of any analysis. Widely applied sample preparation techniques are solid-phase extraction (SPE) and liquid-liquid extraction (LLE).1,2 Both techniques rely on the distribution of the analyte between a donor phase or sample solution and an acceptor phase. For instance, in SPE the sample solution is brought in contact with a solid stationary phase. Due to different partitioning of the analyte with the stationary phase, the analyte can be retained selectively and therefore separated from the bulk solution. After elution of the analyte from the stationary phase, the eluate is evaporated and reconstituted with a suitable diluent. The partition or distribution coefficient of the analyte with the acceptor phase is the driving force but also the limiting factor in SPE as well as in LLE. For example, efficient extraction of polar or ionisable analytes which are more soluble in aqueous phases is challenging in LLE and SPE. Thus, the application of an electrical field as a driving force having orthogonal characteristics to adsorption or partition has attracted interest and this approach has been applied to enhance the extraction of ionisable molecules from aqueous samples.3–15 Selective enrichment of either cationic or anionic molecules can also be accomplished and samples produced from these procedures do not normally require further processing and are often analysed directly. Herbicides are used to control or eliminate unwanted plant growth. They are used particularly in agriculture to increase productivity. Selective herbicides kill specific targets, whereas the non-selective group destroys all plants in contact with them. Paraquat and diquat belong to the latter class and are prominent examples of the family of cationic quaternary ammonium herbicides. Difenzoquat is in the same family, but is applied as selective weed killer.16,17 Other examples of selective herbicides are the anionic mono-, di- and tri- Page 4 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 chlorophenoxy propionic acids, commonly referred as mecoprop, dichlorprop and fenoprop. Mecoprop and dichlorprop are widely used as weedkillers in households and agriculture. Together, they are among the most widely used herbicides in the world and are classified with slight to high toxicity. These anionic and cationic herbicides are also highly polar and thus can easily contaminate aquatic ecosystems.18,19 Analytical methods capable of detecting low concentrations in large volume water samples require dedicated sample preparation procedures. The different polarities and charges of these herbicides would normally require the use of two parallel SPE or LLE experiments, where one experiment would target the anionic analytes and the other would target the cationic analytes. For example, SPE sorbents containing weak cation- or anionexchange properties to enrich the anionic and cationic herbicides, respectively, are used commonly. A polymeric SPE sorbent with methanol, dichlormethane and acetronitrile as conditioning and elution solvents has been applied to extract dichlorprop and mecoprop.20 As with most SPE procedures for large volume samples, large amounts of organic solvent and a tedious workflow were also required for enrichment of quaternary ammonium herbicides from drinking water.21 Thus, there is a need to introduce green sample preparation methodologies as alternatives to these accepted approaches. Green analytical chemistry approaches target to increase extraction efficiency and/or minimise environmental impact. Stacking was developed originally for the sole purpose of increasing the detection sensitivity of CE by increasing the sample load, however stacking is also a means of sample preparation which is open for further exploration since it can tremendously concentrate analytes into a narrow zone.22 We have previously communicated an off-line sample preparation scheme based on stacking by field-enhanced sample injection and demonstrated its use for the selective electrophoretic concentration of anionic analytes.23 The analytes from a large volume of sample were enriched into a microliter volume of higher conductivity Page 5 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 electrolyte that was immobilised inside a micropipette using a hydrogel. Significant analyte enrichment without the use of organic solvents and with limits of detection (LOD) as low as 1 ng/mL after capillary electrophoresis (CE) was obtained. In this work, a new approach to electrophoretic concentration was developed using two micropipettes for the simultaneous concentration and separation of cationic and anionic analytes. The throughput of the approach was increased by implementation in an 8-channel device. Fundamental parameters on this sample preparation called SECS (simultaneous electrophoretic concentration and separation) were investigated using cationic (paraquat, diquat, and difenzoquat) and anionic (mecoprop, dichlorprop and fenoprop) herbicides. SECS was compared to state-of-the-art SPE in terms of analytical performance and environmental considerations. SECS was then optimised and applied to the analysis of herbicides in fortified drinking and river water samples in combination with analysis using high performance liquid chromatography (HPLC) and CE. Page 6 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6
EXPERIMENTAL
Reagents and stock solutions
Acrylamide (>99%), potassium persulfate (>99%), ammonium acetate (>99%-wt), sodium phosphate monobasic (NaH2PO4, >99%), methanol (HPLC grade), acetonitrile (HPLC grade), formic acid (>95%), acetic acid (>99.7%), phosphoric acid (85%), methylene blue), and Ponceau 4R were obtained from Sigma-Aldrich (New South Wales, Australia) and used as delivered. Purified water was from a Milli-Q system (Millipore, MA, USA). Stock electrolyte solutions of 1 mol/L sodium phosphate pH 2.4 and 0.5 mol/L ammonium acetate pH 9 and pH 5 were prepared in purified water. The pH of the stock solutions was adjusted when needed using 1 mol/L sodium hydroxide or 30% ammonium hydroxide solution. The pH and conductivity of solutions were measured using a bench top meter from Sper Scientific (Australia). All stock solutions were sonicated and filtered using a 0.45 µm filter prior to use. Drinking water was collected from a tap and river water from the Derwent River (New Norfolk, Tasmania, Australia). Paraquat tetrahydrate (99.5%) and difenzoquat methylsulfate (99.5%) were purchased from Supelco (Bellefonte, PA, USA) and diquat monohydrate (99.9%), 2-(4-chloro-2methylphenoxy)propionic acid (99.6%, mecoprop), 2-(2,4-dichlorophenoxy)propionic acid (99.9%, dichlorprop) and 2-(2,4,5-trichlorophenoxy)propionic acid (97.7%, fenoprop) were obtained from Fluka Analytical (St. Louis, MO, USA) and used without further purification. Analyte stock solutions of 10 mg/mL each in purified water or 50% acetonitrile were prepared and stored at 5-8°C when not in use. The stock analyte mixture comprised 100 µg/mL of both cationic (paraquat, diquat, difenzoquat) and anionic (mecoprop, dichlorprop and fenoprop) herbicides in purified water. Aliquots of this solution were spiked into the sample solution to make up the final concentration. Page 7 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7
Hydrogel preparation
Hydrogels were prepared directly in 3 mL capacity polypropylene syringes with the plunger removed from and where the narrower end was sealed with parafilm. A basic hydrogel (total volume = 1.2 mL) was made by mixing 600µL 55%-wt acrylamide (monomer), 120 µL 0.5 mol/L ammonium acetate at pH 9, 420 µL purified water, and 60 µL 5%-wt potassium persulfate (initiator). An acidic hydrogel was prepared using the same conditions except with 120 µL 0.5 mol/L ammonium acetate pH 5 instead of pH 9. The mixture was heated at 60oC for 10 min.23 Simultaneous electrophoretic sample concentration and separation (SECS) of cationic and anionic herbicides The scheme for SECS is depicted in Fig. 1 and a diagram showing the SECS procedure is found in Supporting Information (SI) Fig. S1. Two micropipettes were filled with a solution of ammonium acetate at pH 9 (the acceptor phase for anionic herbicides) or pH 5 (the acceptor phase for cationic herbicides) (SI Fig. S1a). The micropipettes containing the acceptor phases were then partially inserted into the bottom of the basic and acidic hydrogels, respectively, contained in the disposable syringe barrels. A platinum wire was attached at the top of each of the hydrogels. The micropipettes were then dipped into the same sample solution containing anionic and cationic analytes (SI Fig. S1b). The sample volume was kept at 20 mL. Voltage was applied with positive electrode at the basic hydrogel and negative electrode at the acidic hydrogel (SI Fig. S1c). This produced an electric field to attract the negatively and positively charged herbicides into the pH 9 (red) and pH 5 (blue) acceptor phases inside the micropipette, respectively. The net liquid flow inside the micropipette was zero because of the hydrogel.24 After application of voltage for the desired time, the entire acceptor solutions were then transferred into separate vials (SI Fig. S1d). The Page 8 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 pH 5 acceptor phases containing the cationic herbicides were analysed by CE. The pH 9 acceptor phases with the anionic herbicides were analysed by HPLC. This experimental set-up was expanded for the simultaneous analysis of up to eight samples with an 8-channel high voltage power supply HVS448-6000D from Labsmith (Livermore, CA, USA) that was capable of providing adjustable voltages of -3 to 3 kV (1 V increments). This expanded set-up is shown in SI Fig. S2. The 20 µL micropipettes (length = 6.4 cm and an inner diameter = 0.3 mm) were from Drummond Scientific Company (Broomall, PA, USA) , 3 mL disposable plastic syringes were from Terumo (Binan, Laguna, Philippines), and 50 mL capacity polypropylene sample vials were from Sarstedt (Mawson Lakes, SA, Australia). The 10-place magnetic stirrer was from LabCo (Cambridge, TAS, Australia) with stirrer bars (length x width = 3 x 1 mm). The applied voltage was adjusted based on the conductivity of the sample matrix (2.7, 63.5, and 112.3 µS/cm for purified, drinking and river water, respectively). As a system suitability test, the applied voltage was adjusted such that the observed current was 200–300 µA after 1 min. This avoided bubble formation inside the micropipette which was observed when the current was >400 µA. The applied voltage for purified, drinking and river water was 2.0, 1.0, and 0.5 kV, respectively.
Weak ion-exchange SPE
Weak cation-exchange of cationic herbicides A 12-port vacuum manifold (Visiprep) from Sigma-Aldrich (NSW, Australia) was used for parallel ion-exchange SPE. Cationic herbicides were extracted using polymeric weak cation-exchange SPE cartridges with 1g sorbent mass and 12 mL volume (Strata-X-CW Page 9 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9 33u) from Phenomenex (NSW, Australia). The manufacturer’s recommended procedure was modified because of the poor concentration factors and repeatability for the highly polar paraquat and diquat. In order to effectively enrich the analytes into the sorbent, a lower flow rate and lower concentration of washing buffer were implemented. The following steps (for one SPE cartridge) were found to be optimal. The sorbent bed was conditioned with 20 mL acetonitrile and then equilibrated with 20 mL 1 mmol/L sodium phosphate pH 7. 20 mL sample solution consisting of 1 mmol/L sodium phosphate pH 7 was loaded at a flow rate of 2 mL/min. The sorbent was then washed with 20 mL 1 mmol/L sodium phosphate at pH 7 and 20 mL 50% methanol. Finally, elution was at 2 mL/min using 20 mL 5% formic acid in acetonitrile into 50 mL centrifuge tube from Nest (China). The eluates were completely dried overnight at 60oC and 550 mbar and reconstituted in 1 mL 50 mmol/L ammonium acetate pH 5. Weak anion-exchange of anionic herbicides Anionic herbicides were extracted using polymeric weak anion-exchange SPE cartridges with 1g sorbent mass and 12 mL volume (Strata-X-AW 33u) from Phenomenex (NSW, Australia). The procedure recommended by the manufacturer was found to be suitable and was as follows. The sorbent bed was conditioned with 20 mL acetonitrile and then equilibrated with 20 mL of 5 mmol/L ammonium acetate pH 6.2. 20 mL sample solution buffered with 5 mmol/L ammonium acetate pH 6.2 was loaded at a flow rate of 2 mL/min. The sorbent was washed with 20 mL of 5 mmol/L ammonium acetate at pH 6.2 and 20 mL methanol. Finally, the anionic herbicides were eluted at 2 mL/min using 20 mL 5% ammonium hydroxide in acetonitrile. The eluates were completely dried overnight at 60 oC and 550 mbar and reconstituted in 1 mL 50 mmol/L ammonium acetate pH 9. Page 10 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10 Analysis of standards, samples and concentrates CE and HPLC were used to analyse the cationic and anionic herbicides, respectively. CE was performed on a G1600 Agilent 3D system (Waldbronn, Germany). Fused-silica capillaries (33 cm total length, 24.5 cm to the detection window) were obtained from Molex (Phoenix, AZ, USA). The separation electrolyte was 150 mmol/L sodium phosphate at pH 2.4 and the applied voltage was -10 kV, respectively. The capillary was thermostated at 20oC, detection was by UV-absorption at 200 nm, and the sample injection was at 50 mbar for 5 s. The electrophoretic mobility of the herbicides were determined using CE. For the cationic herbicides, the separation electrolyte was 50 mmol/L ammonium acetate at pH 5 and the separation voltage was 20 kV. For the anionic herbicides, the electrolyte was 50 mmol/L ammonium acetate at pH 9 and voltage was -20 kV. The mobility values for paraquat, diquat, difenzoquat, dichloprop, mecoprop and fenoprop were 4.7 x 10-08, 4.6 x 10-08, 1.9 x 10-08, -2.1 x 10-08, -2.1 x 10-08, and -2.0 x 10-08 m2/V s, respectively. HPLC was performed on an Agilent 1200 Infinity system (Waldbronn, Germany) using a Dionex Acclaim 120 column (C18, 5µm, 120Ǻ, 4.6 mm diameter and 150 mm length) thermostated to 25 oC. The isocratic mobile phase consisted of 64%-v/v of 25 mmol/L NaH2PO4 at pH 2.9 and 36%-v/v acetonitrile. The flow rate, injection volume and UV detection was 1 mL/min, 10 µL and 230 nm, respectively. Page 11 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 11
RESULTS AND DISCUSSION
Optimisation of SECS The effect of stirring and electrolyte concentration in the acceptor phases were evaluated using purified water as the sample diluent for the herbicides. The acceptor phases were 50 mmol/L ammonium acetate at pH 5 and pH 9. Voltage was at 2 kV for 30 min without and with stirring at 600 rpm. A higher stirring rate was avoided, because this created a vortex. The concentration factor for each analyte is shown in SI Fig. S3. The experiments with stirring provided higher factors which were 150-300 and 77-94 for the cationic and anionic herbicides, respectively. Without stirring the concentration factors were 57-106 and 76-83, respectively. The concentration factors were also repeatable when stirring was applied. With stirring, the %RSD values of concentration factors were 5.3-7.0% and 7.8- 12.0% for the anionic and cationic herbicides, respectively. The %RSDs without stirring were 36.2-88.5% and 25.2-29.4%, correspondingly. Therefore, stirring is critical to the success of SECS. This was also in agreement with the results from other groups working on electric field driven sample concentration.12,25 For example, Hirokawa and co-workers observed enhanced transport of analytes in electrokinetic supercharging-CE by sample stirring.25 Without stirring, analyte depletion was observed visually with the aid of high mobility cationic (methylene blue) and anionic (Ponceau 4R) dyes) in the top half of the sample solution (data not shown). Thus, the analytes were only accessible for SECS in this top half of the sample. In electrokinetic injection, which is the basis of SECS, there is greater preference for higher mobility ions to enter the pipette. This is also known in CE as the bias occurring with electrokinetic injection.26 With stirring, the entire sample solution was accessible to electrokinetic injection, thus larger amounts of the high mobility herbicides Page 12 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 12 were introduced. The injection of lower mobility analytes (all anionic herbicides and difenzoquat) was slow and thus the amount of herbicides accessible to SECS was not significantly different with or without stirring. This also explains the different concentration factors observed for the herbicides in SECS with or without stirring. The concentration of ammonium acetate in the pH 5 and pH 9 acceptor phases was studied at 25, 50 and 100 mmol/L. The concentration of ammonium acetate was the same in both anion and cation acceptor phases during each SECS experiment. Voltage and voltage application time were 2 kV and 30 min. The concentration factor was calculated by dividing the peak area of the sample obtained after sample preparation with the peak area of a standard and then multiplied with the dilution factor. The concentration factors using an acceptor concentration of 25 mmol/L were 59-86 and 59-79 for the cationic and anionic herbicides, respectively. The factors using 50 mmol/L were 150-337 and 18-24 and using 100 mmol/L were 116-190 and 10-12, respectively. Given the above conditions for voltage and voltage application time, the 50 mmol/L acceptor phases were selected for further studies, because they provided the highest averaged concentration factors. In addition, the 50 mmol/L acceptor phases provided a lower running current which offered better stability. The amount of sample ions concentrated into the acceptor phase depends on the electrophoretic concentration time and the nature of the water sample.23 The voltage application time in SECS for purified, drinking, and river water was then studied using 50 mmol/L ammonium acetate at pH 5 and 9 as acceptor phases and with sample stirring at 600 rpm. The results are shown in SI Fig. S4(a), (b), and (c), respectively. For purified water, the concentration factors of the higher mobility cations (paraquat and diquat) reached a maximum at 30 min. The factors then decreased when the time was >30 min, because a Page 13 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 fraction of the concentrated cations migrated out of the micropipette into the hydrogel. For the lower mobility cations and anions (difenzoquat, mecoprop, dichlorprop and fenoprop), the concentration factors gradually increased over 90 min and 60 min, respectively. This was in agreement with electrophoresis, where the concentrated zones of the high mobility ions migrated at higher velocity than the low mobility ions. For the drinking and river water samples, the analytes also reached a maximum concentration factor which decreased as the concentration time was increased. The observed lower concentration factors compared to purified water resulted because the other ions in the sample also entered the acceptor phase. The voltage application time for all the water samples was selected to be 30 min which provided acceptable concentration factors for all analytes.
Environmental considerations (of SECS and SPE)
According to the 12 principles of Green Chemistry and their transformation into analytical chemistry, the main points of concern were the time required and the significant volume of organic solvents used for sample preparation.27–29 In the SPE of charged herbicides, there were seven steps from conditioning of the cartridge through to elution and reconstitution. For instance, one sample preparation cycle for the anionic herbicides involved the use of 59 mL organic solvents and 41 mL aqueous buffer. The analytes were eluted from the SPE cartridge and the eluate was evaporated to dryness overnight. Evaporation was the most time-consuming step which accounted for more than 12h. A similar procedure was performed for the cationic herbicides and involved the use of 49 mL organic solvents and 51 mL aqueous buffer. SPE for both groups of herbicides was accomplished simultaneously. Therefore, the total sample preparation time was approximately 12 h 40 min and the total Page 14 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 14 solvent volume was 200 mL (54% was organic solvent). The cost of two SPE cartridges was approximately 30 AUD (15 AUD each). On the contrary, an SECS cycle for both groups of herbicides took less than 45 min. Four simple steps were involved and these did not require the use of any organic solvents. The total volume of aqueous solution was approximately 2.4 mL. These translate to a reduction in solvent/reagent and time of 99% and 94%, respectively. Additional advantage of SECS was the reduction in running costs. The cost of two micropipettes and two plastic tubes was less than 0.9 AUD. Analytical figures of merit applied in quality assurance/quality control of the entire procedure Analytical figures of merit for the entire analytical procedure (sample preparation by SPE or SECS and analysis by HPLC or CE) were determined. The procedures for cations were CE analysis of the reconstituted extract from weak cation-exchange SPE and the cationic concentrate from SECS. The procedures for anions were HPLC analysis of the reconstituted extract from weak anion-exchange SPE and the anionic concentrate from SECS. The parameters used were the calibration range including regression line, squared correlation coefficient (R2), method detection limit (MDL), method quantitation limit (MQL), precision expressed as repeatability and intermediate precision as well as uncertainty associated with repeatability (U).30,31 Linearity and MDLs were obtained by adding the herbicide stock solution to purified water in the range of 10-2000 ng/mL (each concentration was analysed in duplicate). Then, SECS and SPE were performed followed by quantification of the concentrate and reconstituted extract, respectively. The MDLs were calculated at a signal-to-noise ratio (S/N) of 3 based on the electropherogram and chromatogram obtained from the entire procedure. MQLs were Page 15 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 15 calculated as 3.3x MDL. Precision was evaluated through the percentage relative standard deviation (%RSD) of repeatability and intermediate precision. A sample solution with a concentration of 500 µg/mL of each herbicide was used as repeatability sample. Repeatability was calculated from analysis of 6 sample treatments during one day (%RSD, n=6). Intermediate precision was calculated from analysis of 6 sample treatments, 3 treatments per day for 2 days (%RSD, n=6). Uncertainty associated with repeatability (U) was calculated according to equation (1) = ∗ √ (1) where k is the coverage factor (k = 2 at 95% confidence interval), SD is the standard deviation, and n is the number of measurements. Comparison of analytical figures of merit (SECS and SPE) Table 1 shows the analytical figures of merit and concentration factors for the analysis of cations and anions by CE and HPLC, respectively, with sample preparation by (a) SECS and (b) SPE. The linear range for both sample preparation techniques combined with HPLC or CE was similar and was approximately two orders of magnitude. In terms of sensitivity, the analytical procedures were comparable. However, electrophoretic concentration of the cationic herbicides provided MDLs that were 10-times lower than for the same analytes extracted by SPE. This resulted from the fast migration of the cationic herbicides into the micropipettes and therefore higher enrichment factors. In SPE, the enrichment is limited by the capacity of the sorbent and the sample loading, and volume after reconstitution. The repeatability of the analytical procedure with SECS was 2.4 - 8.8%. The reportable values which considered the calculated U from the analysis of the repeatability sample (500 ng/mL each) for paraquat, diquat, difenzoquat, dichlorprop, mecoprop, and fenoprop were 500 ± 10, 500 ± 22, 500 ± 36, 500 ± 12, 500 ± 20, 500 ± 19 ng/mL, Page 16 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 16 respectively. With SPE, the repeatability values were slightly better at 1.7 - 7.5%. The reportable values were 500 ± 13, 500 ± 31, 500 ± 15, 500 ± 9, 500 ± 26, 500 ± 7 ng/mL, correspondingly. The intermediate precision was also slightly better with SPE (2.6 - 10.0%) compared to SECS (3.0 - 12.0%). The maximum value for concentration factor is the volume of the sample solution divided by the volume used for reconstitution (SPE) or as acceptor phase (SECS). In this work, the maximum values for SECS and SPE are 1000 (= 20 mL/20 µL) and 20 (= 20 mL/1 mL), respectively. Higher concentration factors for SPE could be obtained by increasing the sample volume and/or decreasing the volume for reconstitution. The latter was difficult and produced poor repeatabilities and thus is generally not applicable. The sample volume was increased to 1000 mL in order to have the same maximum factor of 1000. However, it took more than 8 h (at a flow rate of 2 ml/min) to load the sample in the SPE cartridge. The actual concentration factors for SECS were 150 - 337 and 18 - 24 for the cationic and anionic herbicides, respectively. For SPE, the concentration factors were 16 - 20 and 17 - 18 for the cationic and anionic herbicides, correspondingly. SECS is a non-exhaustive technique since it is extremely difficult to transfer all the analytes from a large volume of sample into a small volume of acceptor phase in a single step and within a short period of time. However, the large volume ratio allowed higher concentration factors (larger than one order of concentration magnitude) compared to SPE. Application of SECS to drinking and river water The analytical figures of merit and concentration factors are shown in SI Table S1. The %RSD for repeatability/intermediate precision of drinking and river water were 2.1 - 8.8%/6.1 - 10.3% and 3.2 - 9.1%/5.8 - 12.1%, respectively. The concentration factors were 32 - 124 and 31 - 83, respectively. The correlation coefficients (R2) for calibration linearity Page 17 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 17 for drinking and river water samples were acceptable at 0.997 - 1.000 and 0.998 - 0.999, respectively. MDLs and MQLs were 5 and 17 ng/mL, respectively, for both sample matrixes. The health-related guideline values in Australia for drinking water for paraquat, diquat, difenzoquat, mecoprop, dichlorprop, and fenoprop are 20, 7, 100, 10, 100, and 40 ng/mL.32 Thus, SECS could easily be used for drinking water monitoring. The use of an internal standard or standard addition methods is a possible strategy for accurate analyte concentration determination. The concentration factors behaved differently for drinking and river water compared to purified water. The values were highest for cationic herbicides in purified water. For the complex water samples, the values were highest for the anionic analytes. This reversal behaviour in concentration factor is attributed to the sample matrix. The presence of high mobility cations in drinking and river water samples reduced the efficiency of electrokinetic injection for the cationic herbicides. In SECS, ions from the acceptor electrolyte are replaced by ions from the sample. The high mobility cations in the sample competed with the target analyte ions. Page 18 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18
CONCLUSIONS
Sample stacking which was originally used to overcome the poor detection sensitivity in CE was applied here as a simple, fast and environmentally-friendly sample preparation method. Cationic and anionic analytes were simultaneously concentrated and separated using a simple set-up in a sample preparation device capable of 8 parallel experiments. With the use of an electric field and a large volume ratio between sample and acceptor phase, significant concentration factors in a short period of time were obtained. No organic solvents were involved and the concentrate was directly compatible with liquid phase analytical separation techniques. The method (entitled SECS) compared to SPE was also easier to optimise and gave larger concentration factors for some analytes. From the environmental point of view, SECS was a faster and greener sample preparation technique for large volume aqueous samples. The approach could easily be applied to other charged samples or adopted by other chemists anywhere. The potential of this approach is currently being investigated for the analysis of drugs (tricyclic antidepressants, β-blockers, penicillins, etc.), pesticides (glufosinate, glyphosate) and other bioactive compounds in environmental as well as food samples (e.g., juices, beer and alcoholic beverages). The use of other chemical analysis techniques such as mass spectrometry is also being explored.
ACKNOWLEDGEMENT
JPQ is supported by the Australian Research Council (FT100100213). AW is supported by an International Postgraduate Scholarship of the University of Tasmania. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org/. Page 19 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 Page 20 of 26 ACS Paragon Plus Environment Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20
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