Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES
Technical Note
Automated Agitation-Assisted Demulsification Dispersive Liquid-Liquid Microextraction Liang Guo, Shaohua Chia, and Hian Kee Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03919 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 3, 2016
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.
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.
Page 1 of 18
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
Analytical Chemistry
Automated Agitation-Assisted Demulsification Dispersive Liquid-Liquid Microextraction
Liang Guo,a,b Shao Hua Chia,a Hian Kee Lee*,a,b
a
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore
117543 b
National University of Singapore Environmental Research Institute, T-Lab Building #02-01, 5A
Engineering Drive 1, Singapore 117411, Singapore
*Corresponding author. Email:
[email protected] Tel.: +65 6516 2995; fax: +65 6779 1691
1
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
ABSTRACT Dispersive liquid-liquid microextraction (DLLME) is an extremely fast and efficient sample preparation procedure. To fully exploit its capability and applicability, full automation of its operations seamlessly integrated with analysis is necessary. In this work, for the first time, fully automated agitation-assisted demulsification (AAD)-DLLME, integrated with gas chromatography/mass spectrometry, was developed for the convenient and efficient determination of polycyclic aromatic hydrocarbons in environmental water samples. The use of a commercially available multipurpose autosampler equipped with two microsyringes of different capacities, allowed elimination or significant reduction of manpower, labour and time with the large-volume microsyringe used for liquid transfers, and the small-volume microsyringe for extract collection and injection for analysis. Apart from enhancing accessibility of DLLME, the procedure was characterized by the application of agitation after extraction, to break up the emulsion (that otherwise would need centrifugation or a demulsification solvent), further improving overall operational efficiency and flexibility. Additionally, the application of lowdensity solvent as extractant facilitated the easy collection of extract as the upper layer over water. Some parameters affecting the automated AAD-DDLME procedure were investigated. Under the optimized conditions, the procedure provided good linearity (ranging from a minimum of 0.1-0.5 µg/L to a maximum of 50 µg/L), low limits of detection (0.010-0.058 µg/L), and good repeatability of the extractions (relative standard deviations, below 5.3%, n=6). The proposed method was applied to analyze PAHs in real river water samples.
2 ACS Paragon Plus Environment
Page 2 of 18
Page 3 of 18
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
Analytical Chemistry
INTRODUCTION Sample preparation or extraction often plays a decisive role in an analytical process due to its direct effects on the final results.
1,2
Current efforts in the development and improvement of
extraction process are trending towards microextraction, characterized by solvent minimization, high efficiency, environmental friendliness, and automation, amongst other benefits.3 Dispersive liquid-liquid microextraction (DLLME), first introduced by Rezaee et al.2 in 2006, is an outstanding example of the many solvent-minimized extraction methods developed in the past twenty years. In DLLME, by rapid injection into the aqueous sample solution together with a water-miscible dispersive solvent, the extraction solvent becomes highly dispersed in the form of fine droplets into which the analytes could be extracted very rapidly.4 Featuring speed, easy operation, and reduced consumption of organic solvent, DLLME, since its introduction, has significant application. 5-12 In the most basic versions of DLLME, denser-than-water organic extraction solvents are used to facilitate the collection of the final extract sedimentated by centrifugation. This has limited the applicability of the procedure since there are more low-density solvents consider for liquid-based extractions. Several studies have reported the use of low-density solvents in DLLME. 13-24 Automation is an important developmental trend in sample extraction. However, the exploration of automated DLLME has been scarce so far since the separation of the final extractant usually requires centrifugation. Nevertheless, there have been some attempts to automate DLLME, such as on-line sequential injection based DLLME,25-27 and automated insyringe DLLME.28,29 However, the use of such in-house-assembled setups limits their accessibility by the scientific community, and also adds operational complexity.30 Most reports do not make it clear if the automation described therein applied to the consideration of single samples only, or of multiple samples consecutively and uninterruptedly. “Full automation” is, of course, represented by the latter. In our previous work,31 we reported a fully automated DLLME procedure, using a low density solvent as the extraction solvent to facilitate the convenient collection of the upper layer extract. However, since a single syringe was used on an autosampler to carry out all the automated steps, the procedure was a compromise between the fast and more efficient transfer of 3 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
solvent (for which a large volume syringe was preferred), and the convenient collection of a small volume of the final extract (in which a lower volume syringe would be optimal). Thus, since GC/MS performance was considered relatively more important, the lower-volume syringe was selected. This, however, affected the speed and efficiency of solvent transfers. Additionally, because a demulsification solvent was used to clear the emulsion, the amount of solvent consumption was relatively large. In the present work, a dual-syringe based automated agitation-assisted demulsification DLLME (AAD-DLLME) procedure was developed. A large volume microsyringe was used in the DLLME steps to transfer solvents to speed up the process, while a small volume microsyringe was used to collect the extract conveniently and inject it into the GC/MS system. Agitation was used to clear the emulsion to avoid the use of demulsification solvent. The main parameters affecting DLLME were studied and optimized. The proposed method was applied to determine polycyclic aromatic hydrocarbons (PAHs) in genuine river water samples.
EXPERIMENTAL SECTION Chemicals and Materials. PAH (Kit 610-N) standards (acenaphthene (Ace), acenaphthylene (Acp), anthracene (Ant), benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), chrysene (Cry), dibenz[a,h]anthracene (DBA), indeno[1,2,3cd]pyrene (InP), fluoranthene (Flt), benzo[g,h,i]perylene (BghiP), fluorene (Flu), naphthalene (Nap), phenanthrene (Phe), and pyrene (Pyr)) were bought from Supelco (Bellefonte, PA, USA). The details of solvents and chemicals used are provided as Supporting Information. Sample Preparation. The details of preparation of stock solution and water samples used to study extraction performance, and the collection of genuine water sample are provided as Supporting Information. Instrumentation. A Gerstel (Mülheim an der Ruhr, Germany) two-rail multipurpose sampler (MPS), coupled to a GC/MS system, was used to conduct the automated AAD-DLLME procedure. An orbital agitator was equipped as part of the MPS. A Gerstel 250 µL GC microsyringe affixed to the autosampler was used for the extraction operations and a Gerstel 10 µL GC microsyringe was used for collecting and injecting the final extract into the GC/MS 4 ACS Paragon Plus Environment
Page 4 of 18
Page 5 of 18
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
Analytical Chemistry
system. The in-house-designed and modified glass vials with narrow necks used in the automated procedure were modified from conventional vials (details are provided as Supporting Information).31 The details of GC/MS analysis are provided as Supporting Information. Automated Agitation-Assisted Demulsification DLLME. The entire DLLME procedure including the solvent mixture injection, the breakup of the emulsion by agitation, the collection of the extract as the upper layer, and the introduction of the final extract into the GC/MS system, was carried out on the Gerstel MPS, simultaneously equipped with a 250 µL GC microsyringe on the left rail and a 10 µL GC microsyringe on the right. The optimized automated AAD-DLLME procedure was as follows (Figure 1), 160 µL of mixture of hexane (as extraction solvent) and acetone (as dispersive solvent) (1:3, v/v) was withdrawn into the 250 µL microsyringe, which was moved to the sample vial, to inject the mixture into the sample solution rapidly. A cloudy mixture consisting of the extraction solvent, the dispersive solvent and the aqueous sample was formed due to the highly dispersed extraction solvent in the aqueous phase. After extraction, the sample vial was transported to the agitator and agitated at 600 revolutions per minute (rpm) for 10 min for phase separation, and then transported back to the tray. The transportation steps were also carried out by the left rail. At this juncture, the organic solvent (extract) represented the upper layer of the solution. One milliliter of ultrapure water was added into the sample vial via the left-rail microsyringe; this made the lighter than water organic solvent layer to be elevated into the narrow neck of the sample vial for convenient collection. One microliter of the extract was then collected by the 10 µL microsyringe held at the right rail, and injected into the GC/MS system for analysis. The entire AAD-DLLME procedure was automatically repeated to extract and analyze on the subsequent sample sequentially, and uninterruptedly.
RESULTS AND DISCUSSION The extraction efficiency in DLLME, whether automated or otherwise, is affected by various parameters such as the choice of extraction solvent, the type of dispersive solvent, the respective volumes of both solvents, and duration and speed of agitation. All optimization experiments were performed in triplicate. 5 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
Extraction Solvent Type and Volume. In the automated mode, designed and developed in this work, for convenient collection of the extract as the upper layer after extraction, the solvent should have a lower density than water. Four organic solvents including hexane, toluene, oxylene and 1-octanol were evaluated. From Figure 2, it can be seen that the peak areas obtained by hexane was slightly higher than that obtained by 1-octanol, and observably higher than those obtained by toluene and o-xylene. Thus hexane was chosen as the extraction solvent for subsequent experiments. The volumes of hexane were evaluated, and 40 µL of hexane was deemed to be the most favourable minimized extraction solvent volume (details are provided as Supporting Information). Dispersive Solvent Type and Volume. Three commonly used dispersive solvents (acetone, acetonitrile, and methanol) were evaluated. Based on the peak area obtained, acetone was selected as the dispersive solvent (details are provided as Supporting Information). To determine the influence of the volume of the dispersive solvent on extraction, a series of volumes (80, 120, 160, 200, 400, and 800 µL), was investigated. The peak areas obtained by using 120 µL of dispersive solvent (acetone) were slightly higher than those obtained by using 80 µL and 160 µL, and much higher than those obtained by using other volumes (shown in Figure S-3). With 80 µL, the emulsion did not form very well because the extraction solvent could not be dispersed completely into the aqueous sample solution, leading to lower extraction. On the other hand, the use of relatively higher dispersive solvent volumes, would lead to increased solubility of analytes in the sample solution, and negatively affect the extraction. The same observation has been reported in previous studies.2 Therefore, in subsequent experiments, a volume of 120 µL was deemed to be the most favourable for analyte extraction. Duration and Speed of Agitation. In order to collect the extraction solvent after extraction, agitation was applied to hasten phase separation.12 Different periods of agitation (2, 5, 10, 15, and 20 min) were studied, and the results for 6 selected PAHs are illustrated in Figure 3 (for clarity, details of all 16 PAHs are provided as Supporting Information (Figure S-5 )). It can be seen that the peak areas increased going from 2 min to 5 min, and reached a maximum at 10-15 min, after which the peak areas decreased, especially for lower molecular PAHs. This is because 2 and 5 min of agitation were insufficient for the emulsion to be fully removed. However, a prolonged duration of 20 min conceivably led to the increased distribution of the semi-volatile
6 ACS Paragon Plus Environment
Page 6 of 18
Page 7 of 18
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
Analytical Chemistry
PAHs to the headspace, resulting in the overall loss of extracted analytes. An agitation time of 10 min was thus adopted as the agitation time. The most favourable agitation speed was at 600 rpm (details are provided as Supporting Information). Based on the above discussion, the most favourable extraction conditions for automated AADLLME were as follows: 40 µL of hexane as extraction solvent, together with 120 µL of acetone as dispersive solvent. After extraction, the sample was agitated at 600 rpm for 10 min to affect phase separation. Method Evaluation. Under the optimized extraction parameters, linearity, precision, repeatability, limits of detection (LODs), and limits of quantification (LOQs) were calculated to validate AA-DLLME performance, using ultrapure water spiked with the target analytes as sample solutions. The results are summarised in Table 1. Good linearity of the method was obtained. It ranged from a minimum of 0.1-0.5 µg/L to a maximum of 50 µg/L, with regression coefficients (r2) ≥ 0.9905. The relative standard deviations (RDS, %, n=6) were calculated for the extraction and analysis of spiked water samples at LOQ levels of the analytes to evaluate the precision of the developed method. The RSDs were below 5.3% for all analytes, indicating good repeatability of the method, as expected from the automated process. The LODs for the PAHs based on a signal-to-noise (S/N) ratio of 3, ranged from 0.010 to 0.058 µg/L. The LOQs, based on a S/N ratio of 10, ranged between 0.035 to 0.191 µg/L. The results of LODs obtained by the proposed procedure were lower than or comparable with those of several previously reported DLLME methods for the determination of PAHs, such as low density solvent based DLLME-GC/MS,32
water with low concentration of surfactant in
dispersed solvent-assisted emulsion DLLME-GC/MS,33 and combined subcritical water extraction and DLLME-GC/MS,34 but higher than low toxic (bromo/iodo solvents based) DLLME-GC/MS.35 Compared to other methods, the outstanding feature and advantage of the present procedure is its full automation, spanning consecutive experiments, and not only within single experiments, which provides manual labour-free convenience as well as exploits fully the benefits of DLLME (speed, simplicity, effectiveness, efficiency, and high enrichment). Analysis of Genuine River Water Samples. The developed AAD-DLLME-GC/MS approach was applied to determine PAHs in genuine river water samples to evaluate the real world application of the method. From the results that are summarised in Table 2, it can be observed 7 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
that the concentrations of PAHs ranged from 0.32 µg/L to non-detected. The latter result indicated the absence of the contaminants or their concentrations were below the LODs of the procedure. To assess matrix effects, the genuine river water samples were spiked at 1.0 and 10 µg/L concentrations of each PAH standard, respectively, and were subjected to the procedure. The relative recoveries, calculated from the ratio of the peak areas of the analytes in river water extracts to the peak areas of the analytes in ultrapure water extracts spiked at the same concentrations, are listed in Table 2. The relative recoveries ranged from 85.8% to 107.2%, with RSDs (n=6) of
