Fully automated plunger in needle liquid phase microextraction

FULLY AUTOMATED PLUNGER-IN-NEEDLE LIQUID-PHASE MICROEXTRACTION TIAN YUHAO NATIONAL UNIVERSITY OF SINGAPORE 2014 FULLY AUTOMATED PLUNGER-IN-NEEDLE LIQUID-PHASE MICROEXTRACTION TIAN YUHAO (M.Sc, Peking University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Prof. Lee Hian Kee, Department of Chemistry, National University of Singapore, between August 2013 andAugust 2014. I have duly acknowledged all the sources of information, which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. r t,/ n It' I [ ',r TIAN YUHAO 21 August 2014 ACKNOWLEDGEMENTS I am deeply indebted to Professor Lee Hian Kee, my supervisor at NUS. He has been a great teacher, and I have valued his counsel and guidance. I would like to express my deepest gratitude to my mentor, Dr. Zhang Hong. Work under her guidance was a rigorous learning process. Without her valuable comments and constant quality requirements, my thesis would not have been as it is. There are many people who have contributed to my academic development. I do appreciate very much what I have learnt from my teachers in NUS: Professors Li Fong Yau Sam, Yeo Boon Siang Jason and Lee Hian Kee. They have transmitted to me both knowledge and the passion to do research. I am also grateful to Dr. Zhu Shenfa, and Dr. Brenda Lee for their kind guidence to my written English. I am especially grateful for the help and encouragement I received from all the staff of NUS. The Department of Chemistry, where I have been working, has provided great support for my study. To it, I express my deep gratitude. I am also grateful to the SPORE Project (Singapore-Peking-Oxiford Research Enterprise) and the National Research Foundation for granting me a research scholarship for my study. i Table of Contents ACKNOWLEDGEMENTS ............................................................................. i Table of Contents ............................................................................................. ii SUMMARY ..................................................................................................... iv List of Tables .................................................................................................... v List of Figures .................................................................................................. vi CHAPTER 1 INTRODUCTION................................................................... 1 1.1 Literature review.................................................................................... 1 1.1.1 Introduction of sample preparation ................................................... 1 1.1.2 Introduction of different microextraction methods ........................... 2 1.1.3 Plunger-in-needle liquid-phase microextraction ............................... 7 1.2 Objective and scope of this research .................................................... 9 1.2.1 Research Motivation ......................................................................... 9 1.2.2 Scope of this research ..................................................................... 10 CHAPTER 2 MATERIALS AND METHODS ......................................... 11 2.1 Chemicals and reagents ....................................................................... 11 2.2 Apparatus and instrumentation ......................................................... 12 2.3 Sample solution preparation ............................................................... 12 2.4 Extraction procedure ........................................................................... 13 2.4.1 PIN device preparation ................................................................... 13 2.4.2 Solvent impregnation ...................................................................... 14 ii 2.4.3 Water sampling and extraction ....................................................... 15 2.4.4 Analysis........................................................................................... 17 CHAPTER 3 RESULTS AND DISCUSSION ........................................... 19 3.1 Solvent selection ................................................................................... 19 3.2 Optimisation for solvent impregnation .............................................. 21 3.2.1 Arrangement for preliminary optimisation ..................................... 21 3.2.2 Optimisation of the number of dynamic cycles .............................. 23 3.2.3 Optimisation of agitation speed ...................................................... 26 3.3 Extraction condition optimisation ...................................................... 27 3.3.1 Optimisation of agitation speed ...................................................... 27 3.3.2 Optimisation of flow rate ................................................................ 28 3.3.3 Optimisation of extraction time ...................................................... 30 3.3.4 Desorption temperature and time optimisation ............................... 31 3.4 Application to water samples .............................................................. 33 3.5 Comparison with other microextraction techniques ........................ 35 3.6 Conclusion ............................................................................................ 36 3.7 Future work .......................................................................................... 37 REFERENCES ............................................................................................... 38 iii SUMMARY A novel fully automated continuous-flow plunger-in-needle liquid-phase microextraction (PIN-LPME) technique with gas chromatography/mass spectometric (GC/MS) analysis to determine five organochlorine pesticides (OCPs) from water samples was developed. A peristaltic pump was used to feed water sample from a reservoir into the sample vial. With the utilisation of a CTC CombiPAL autosampler and its associated Cycle Composer software, a sample preparation-GC/MS method was feasible that allowed water sampling, sample extraction, extract injection and analysis to be carried out completely automatically. Optimisation of extraction solvent, solvent impregnation conditions, agitation speed, sampling flow rate and extraction time were carried out successively. The limits of detection for organochlorine pesticides ranged from 0.01 to 0.02 µg/L. The enrichment factors ranged from 108 to 878, with relative standard deviations (RSDs) ranging from 2.8% to 11.9%. This automated continuous-flow PIN-LPME method demonstrated the feasibility of a complete analytical system comprising sampling, sample preparation and GC/MS analysis that might be applied to onsite analysis for environmental samples, automatically. iv List of Tables Table 2-1 Retention time, qualitative ions and quantitive ions of OCPs ......... 18 Table 3-1 Arragement for preliminary optimisation ........................................ 22 Table 3-2 Performance of fully-automated PIN- LPME ................................. 33 Table 3-3 Analysis of genuine water sample spiked at 10 µg/L of each analyte ........................................................................................................ 34 Table 3-4 LOD comparison of fully-automated PIN-LPME with other microextraction techniques (µg/L) ................................................. 35 v List of Figures Figure 1-1 Schematic of SPME manual fibre assembly holder (adapted from Ref. [4]) ............................................................................................ 2 Figure 1-2 Schematic of direct immersion single-drop microextraction (adapted from Ref. [27]) .................................................................. 5 Figure 1-3 Principle of (a) three- and (b) two-phase LPME (adapted from Ref. [36]) .......................................................................................... 6 Figure 1-4 Schematic of the home-assembled PIN-LPME device .................... 7 Figure 1-5 Scanning electron micrographs at different magnifications (left images, 100×; right images, 450×) of the surface of the stainless steel wire before (a and b) and after (c and d) etching [42] (reproduced with permission) .......................................................... 8 Figure 1-6 Illustration of PIN-LPME devices: stages in the preparation of solvent-impregnated hydrofluoric acid-etched stainless steel wire . 9 Figure 2-1 Structures of organochlorine pesticides studied in this report ....... 11 Figure 2-2 Schematic of the PIN device in solvent impregnation step............ 15 Figure 2-3 Schematic of the PIN-LPME device in extraction step ................. 16 Figure 3-1 Comparison of extraction efficiency of five organic solvents and the etched wire without organic solvent for five organochlorine pesticides ........................................................................................ 21 Figure 3-2 Peak areas of 1-octanol under different agitation speeds, immersion time and the number of dynamic cycles ........................................ 23 Figure 3-3 Influence of the number of dynamic cycles under different agitation speed (1, 5 and 15 in this figure stand for the number of dynamic vi cycles) ............................................................................................ 24 Figure 3-4 Comparison of the intensity of solvent under diffrent numbers of dynamice times .............................................................................. 25 Figure 3-5 Signal intensity of 1-octanol under different agitation speed ........ 26 Figure 3-6 Influence of agitation speed on extraction effeciency.................... 28 Figure 3-7 Extraction efficiencies of different analytes under different flow rates (Agitation speed was 600 rpm, extraction time was 20 min) 30 Figure 3-8 Influence of extraction time on extraction efficiency .................... 31 Figure 3-9 Signal Intensity of analytes under different desorption temperature (a) and desorption time (b). ............................................................ 32 Figure 3-10 GC/MS-SIM chromatogram of real samples after fully automated PIN-LPME: spiked reservoir water sample (spiked with 10 µg/L of each OCP). ..................................................................................... 34 vii CHAPTER 1 CHAPTER 1 INTRODUCTION 1.1 Literature review 1.1.1 Introduction of sample preparation Sample pretreatment is one of the most important steps in analytical process. In most cases, samples are complex mixtures of chemicals and only small amounts of them need to be analysed. In addition, most sample matrices are very complex, such as seawater, wastewater, soils, etc. Therefore, the main objectives of sample pretreatment are to simplify the sample matrix and to concentrate the analytes in them. Many sample preparation techniques such as liquid phase extraction and solid phase extraction have been widely used to analyse wastewater, air, soil, and food samples over the years. However, traditional sample pretreatment techniques such as liquid-liquid extraction and liquid-solid extraction have many limitations; they are time consuming, and require manual labor and large volumes of hazardous solvents. Therefore, many improved sample preparation procedures have been developed to meet the requirements of reduced number of preocedural steps, low volumes of solvents in extraction, environmental friendliness, and possiblilities of onsite application, and automation. Among these novel sample preparation techniques that have the potential for meeting all those requirements mentioned above, liquid-phase microextraction (LPME) [1, 2] and solid-phase microextraction (SPME) [3] are the most commonly 1 CHAPTER 1 reported and widely used ones in recent years. 1.1.2 Introduction of different microextraction methods 1.1.2.1 Solid-phase microextraction SPME is a sample preparation technique in which a fused silica or metal fibre coated with a functional coating material is employed to extract analytes from liquid or gas samples. Figure 1-1 shows a schematic of an SPME fibre assembly holder. Figure 1-1 Schematic of SPME manual fibre assembly holder (adapted from Ref. [4]) As reported in many papers, SPME has been applied to air samples [5-9], wastewater samples, biological samples [10], food samples, etc. It has been 2 CHAPTER 1 used for on-site sampling [6, 8, 9, 11]. It is a solvent-less technique, and can be used in both manual operation and complete automation with gas chromatography (GC) or high-performance liquid chromatography (HPLC). Several coatings have been developed for the analysis of environmental pollutants in samples, such as Carbowax 20M-modified silica [12],PDMS– PVA [13, 14], PTMOS and MTMOS [15], LTGC [16, 17], different calix arenes [18], and a variety of crown ethers [19, 20]. SPME has been reported to be a useful sampling device for field investigation for air, water, etc. Although SPME has many advantages, it still has some limitations, such as sample carry-over, fragility of fibres, limited lifetime, relatively expensive costs, polymer decomposition, etc. 1.1.2.2 Liquid-phase microextraction Liquid phase microextraction (LPME) also called solvent microextraction, is commonly defined as a sample pretreatment technique that extracts analytes from gaseous, liquid or solid samples with 100 µL or less volume of solvent [21]. Comparing with traditional liquid phase extraction, LPME is more rapid, convenient and environmentally friendly. LPME techniques have been widely adapted to various sample types and analytes due to its simplicity and low cost, since it was developed in the mid-1990s. There are many operation modes of LPME, such as single-drop microextraction (SDME), hollow fibre-protected microextraction (HF-LPME), and dispersive liquid-liquid microextraction (DLLME). 3 CHAPTER 1 SDME method can be traced back to the work of Liu and Dasgupta in 1995 that a volume of microliter level droplet was used to extract analytes from a gas stream sample[22]. In Jeannot and Cantwell 's work [1], a small droplet located at the end of a Teflon rod was applied to extract 4-methylacetophenone from aqueous sample. SDME has become very popular because it is inexpensive, easy to operate and nearly solvent-free and it can be used in combination with GC, HPLC, ICP and other analytical techniques. Figure 1-2 shows the schematic of direct immersion SDME. In the SDME method, efforts have been made to improve the mass transfer between organic phase and aqueous sample, some operation modes have been developed, such as 1) agitating the aqueous sample, 2) pulling 90% of the drop back into the syringe needle and then pushing it back out repeatedly, which was called in-needle dynamic modes LPME [23], and 3) extracting analytes from a continuous flow of sample solution [24]. SDME can be fully automated using a computer-programmable autosampler, such as a CTC CombiPAL using patented software [25] However, in practical applications, forces generated by stirring of the aqueous sample potentially easily dislodge the microdrop suspended on the needle of microsyringe. Many attempts has been made to deal with this problem, such as a syringe with a beveled needle tip [2], appropriate solvent and a small volume of solvent [26], but they cannot solve this problem completely. 4 CHAPTER 1 Figure 1-2 Schematic of direct immersion single-drop microextraction (adapted from Ref. [27]) HF-LPME was developed by Pederseen-Bjergaard and Rasmussen in 1999. In HF-LPME, the extractant is contained in a porous polypropylene hollow fibre with one end sealed. The hollow fiber protects the extractant from contamination sample matrix [28]. Unlike SDME, the extracting solvent cannot be dislodged and lost. There are two operational modes of HF-LPME, three-phase HF-LPME and two-phase HF-LPME. In the two-phase system, the extractant is in the hollow fibre lumen as well as the pore of the fibre. In the three-phase HF-LPME mode, analytes were extracted into the intermediary solvent phase in the pore of hollow fibre and then subsequently transfered into the aqueous phase in the lumen. HF-LPME has been used to extract 5 CHAPTER 1 pharmaceuticals, pesticides, fungicides, phenols and PAHs from fruit juice, urine-plasma, honey, seawater, wastewater and river water samples [29-35]. Figure 1-3 illustrates the principle of three- and two-phase LPME. Figure 1-3 Principle of (a) three- and (b) two-phase LPME (adapted from Ref. [36]) Dispersive liquid-liquid microextraction (DLLME) makes use of a mixed solution of a water-insoluble extractant with a density higher or lower than water and a water-soluble solvent. The solution was injected into the aqueous to form a stable emulsion. Then, the emulsion was centrifuged to seperate the immiscible edges and the extraction solvent was drawn from the tube with a syringe and analyzed by GC. As reported, DLLME has many applications in sample analysis, such as clozapin in unrine and serum [37], Sudan dyes in egg yolk [38], PAHs in marine sediments [39], hebicides in cereals [40], quercetin in honey [41], etc. 6 CHAPTER 1 1.1.3 Plunger-in-needle liquid-phase microextraction Recently, a novel LPME technique called plunger-in-needle liquid-phase microextraction (PIN-LPME) technique was developed by Zhang and Lee [42]. The schematic of the PIN-LPME device is illustrated in Figure 1-4. Figure 1-4 Schematic of the home-assembled PIN-LPME device The stainless steel plunger wire of a commercial plunger-in-needle microsyringe was etched with hydrofluoric acid to form a microporous structure, and the etched plunger was used as the extractant solvent holder. Figure 1-5 shows the scanning electron micrographs of the surface of the stainless steel wire before and after etching. The extractant could be more easily held within the pores, comparing with the drop in the tip of needle in SDME. When the plunger wire with the extractant was exposed to the sample solution, analytes diffused from the sample solution to the extractant. After 7 CHAPTER 1 extraction, the plunger wire was directly introduced into the injection port of a GC/MS system for analysis of the analytes, which would be vaporied together with the solvent from the plunger. Figure 1-5 Scanning electron micrographs at different magnifications (left images, 100×; right images, 450×) of the surface of the stainless steel wire before (a and b) and after (c and d) etching [42] (reproduced with permission) As reported in Zhang's paper [42], the PIN-LPME showed many advantages that it integrates extraction and extract introduction for analytes into one device The preparation of the etched wire was very convenient, no additional impregnations were required; organic solvent consumption was much reduced; good extraction efficiency, linearity and repeatability were also achieved. As reported, the etched stainless steel wire had good affinity with a variety of organic solvents and underwent no degradation after impregnation 8 CHAPTER 1 of its pores [43]. Figure 1-6 illustrates the hydrofluoric acid-etched stainless steel wire and longitudinal cross-sectional view of solvent-impregnated hydrofluoric acid-etched stainless steel wire. Figure 1-6 Illustration of PIN-LPME devices: stages in the preparation of solvent-impregnated hydrofluoric acid-etched stainless steel wire 1.2 Objective and scope of this research 1.2.1 Research Motivation As described previously, sample preparation is an important part of analytical procedure. With the requirement for environmentally benign, rapid, and convenient sample preparation technique, many microextraction methods have emerged and been widely used, such as SPME and LPME. Reduction in 9 CHAPTER 1 the number of steps, reduction of solvents for extraction, potential adaptability to field sampling, and automation are four main goals for sample preparation improvement. 1.2.2 Scope of this research The novel microextraction technique, PIN-LPME, integrates extraction and extract introduction for analytes into one device and organic solvent consumption was much reduced [42]. Therefore, in this report, 1. A fully automated continuous-flow plunger-in-needle liquid-phase microxtraction (PIN-LPME) system is reported; 2. Several organochlorine pesticides are selected as model analytes to evaluate the procedure; 3. Parameters influencing the impregnation of extractant and the performance of PIN-LPME are investigated and optimized; 4. This system is applied to process environmental water samples. 10 CHAPTER 2 CHAPTER 2 MATERIALS AND METHODS 2.1 Chemicals and reagents 2,4'-Dichlorodiphenyltrichloroethane (2, 4’-DDT; CAS No. 789-02-6), Dieldrin (CAS No. 60-57-1), Heptachlor (CAS No. 76-44-8), 4, 4’-DDT (CAS number: 50-29-3) and HCB (CAS No. 118-74-1, Hexachlorobenzene), were purchased from SPEX CertiPrep (Metuchen, New Jersey, U.S.). The structures of organochlorine pesticides mentioned above are shown in Figure 2-1. Figure 2-1 Structures of organochlorine pesticides studied in this report 11 CHAPTER 2 HPLC-grade 1-octanol, n-hexane, o-xylene and propyl benzoate were purchased from Sigma–Aldrich (St. Louis, MO, USA). Toluene was obtained from Tedia Co. (Fairfield, OH, USA). Ultrapure water was obtained from ELGA Purelab Option-Q (High Wycombe, UK). 2.2 Apparatus and instrumentation Shimadzu QP2010 GC/MS system was purchased from Shimazu (Kyoto, Japan). The CTC Analytics CombiPAL autosampler with an agitator was purchased from CTC Analytics AG, Zwingen, Switzerland. Peristaltic pump and tubings were purchased from Spectra-Teknik, Singapore. The plunger-in-needle syringe with replaceable 26-gauge, 70 mm long needle, 0.47mm internal diameter (I.D.) microsyringe (0.5-µL capacity) was purchased from SGE (Ringwood, VIC, Australia). For LPME applications, a replacement needle (23-gauge, 50 mm long needle, 0.63 mm I.D., SGE) was necessary. The latter one with wider bore and shorter needle allowed the plunger, particularly the solvent-impregnated tip (2.0 cm length), to be withdrawn into it for protection, during PIN-LPME operations, and introduction of the extract into the GC/MS system for analysis [42]. 2.3 Sample solution preparation Organochlorine pesticides (OCPs) are still considered to be priority pollutants to be monitored in many environmental matrices, although the use 12 CHAPTER 2 of OCPs has been discontinued largely as a result of the long history of bioaccumulation, toxicity, and high environmental pesistence. Several OCPs were selected as model analytes to evaluate the fully automated PIN-LPME procedure. Stock solutions of each OCP were prepared in methanol solution at 100 mg/L, individually. All stock solutions were stored in refrigerator at 4°C. Sample solutions were prepared by spiking stock solutions of all analytes into certain volume of ultrapure water daily. As reported, analytes at concentration higher than 50 µg/L in water could be extracted efficiently without addition of salt [25]. As the main purposes of this research are to automate the PIN-LPME and make it suitable for field investigation, no other pretreatment process such as salt addtion was applied. The concentration of each analyte in synthetic samples used in all optimisation studies was 50 µg/L. 2.4 Extraction procedure 2.4.1 PIN device preparation The stainless steel plunger wire was etched to form a rough and porous surface for solvent impregnation. The etching steps were as follows: the plunger wire was cleaned in acetone, wiped with a piece of lint-free tissue and immersed in hydrofluoric acid for 15 min at room temperature. The etched part of the plunger wire was 2 cm long. After etching, the plunger wire was washed gently with ultrapure water and dried under room temperature for 1 13 CHAPTER 2 hour and then conditioned at 300°C in the injection port of the GC for 30 min. 2.4.2 Solvent impregnation In the solvent impregnation step, the etched part was immersed in organic solvent to allow full impregnation of the solvent into the pores. The whole process is presented as follow: The plunger wire was pulled back into the needle. Then the needle was inserted into the solvent vial, which is placed in the agitator. The plunger was pushed out to make the etched wire immersed into the extraction solvent for a certain period of time, which need to be optimised. During the immersion period, the agitator was shaking at a certain speed. After the certain period of time, the agitator was stopped and the plunger was pulled back into the needle. The cycle of steps from the immersion to the pulling back of the plunger were repeated for several times, which need to be optimised. All steps decribed above were manipulated with the CTC Analytics CombiPAL autosampler automatically, which was programmed in the Cycle Composer software. Figure 2-2 shows the schematic of the PIN device during the solvent impregnation process. It was important to ensure that the volume of extraction solvent was not only consistent but also sufficient to give satisfactory extraction efficiency of the target compounds. To obtain a consistent layer of organic solvent on the plunger wire, the wire should be removed immediately from the organic solvent after impregnation [44]. This was essential, as a consistent volume of 14 CHAPTER 2 1-octanol would lead to both high precision and reproducibility of extraction. Figure 2-2 Schematic of the PIN device in solvent impregnation step Parameters to be optimised in solvent impregnation step included solvent selection, the time of immersion, the speed of agitation, and the number of cycles. 2.4.3 Water sampling and extraction After solvent impregnation, the wire was removed and placed in the sample solution for extraction. Considering the application potential for field sampling, a peristaltic pump with two pump heads was used for water sampling. After the solvent impregnation process, the plunger wire was withdrawn into the needle for protection and the assembly was removed from the solvent vial. The needle was then placed in the sample vial, and the etched 15 CHAPTER 2 plunger wire impregnated with extraction solvent was pushed out of the needle and exposed to the sample for the extraction process to begin. Figure 2-3 illustrates the whole extraction process. Figure 2-3 Schematic of the PIN-LPME device in extraction step As shown in Figure 2-3, the continuous flow was formed by pumping the water sample from the reservoir into and out of the sample vial during the extraction process. After extraction for a certain time under a certain agitation speed, the plunger wire was withdrawn into the needle for protection, and the assembly was removed from the sample. 16 CHAPTER 2 The extract was then introduced to the GC/MS system for analysis by piercing the GC injection port septum with the needle and exposing the wire for thermal vaporation of the analytes at 295°C for 10 min. The etched wire could be used repeatedly without deterioration of the analytical results. Here, one single wire was used for all experiments, unless otherwise stated. To prepare the wire for the next experiment, it was left in the GC injector port for another 10 min to remove all trace of the analytes. In the extraction step, there were several parameters to be optimised, such as flow rate, agitation speed and agitation time. 2.4.4 Analysis Analysis was carried out using a Shimadzu QP2010 GC/MS system (Kyoto, Japan) and a DB-5MS fused silica capillary column (30 m, 0.25 mm i.d., 0.25 µm film thickness) (J&W Scientific,Folsom, CA). High purity (99.999%) helium was used as the carrier gas at a flow rate of 1.5 mL/min. The injector temperature was kept at 295°C and operated in the splitless mode. A deactivated single gooseneck splitless inlet linear (3.5 mm i.d., 5.0 mm o.d., 95 mm length) without glass wool from Restek Corporation (Bellefonte, PA) was used. The total flow rate was set at 40.0 mL/min. The MS system was operated in the electron impact ionization mode, and the interface temperature was set at 295°C. The GC temperature program was as follows: initial temperature 50°C, held for 2 min; increased by 30°C /min to 290°C and held for 2 min. A mass range of m/z 50 - 500 was scanned to confirm the retention 17 CHAPTER 2 times of the analytes For each analyte, three fragment ions were selected as qualitative ions to obtain high selectivity, and the most abundant fragment of each analyte was selected for quantification. Table 2-1 shows retention time, qualitative ions and quantitive ions information of five analytes. Table 2-1 Retention time, qualitative ions and quantitive ions of OCPs Analyte Retention time Qualitative ions Quantitive ions (min) HCB 8.366 284, 288, 142 284 Heptachlor 9.147 100, 272, 65 100 Dieldrin 10.074 79, 263, 277 79 2, 4' - DDT 10.300 235, 165, 199 235 4, 4' - DDT 10.553 235, 165, 199 235 All standard and sample solutions were analyzed in GC/MS selective ion monitoring (SIM) mode at least in triplicate. 18 CHAPTER 3 CHAPTER 3 RESULTS AND DISCUSSION 3.1 Solvent selection The selection of solvent as the extraction phase is an important step in LPME to achieve good selectivity and efficient enrichment. In the PIN-LPME, the organic solvent was filled in pores of etched plunger when the plunger was immersed in the solvent and subsequently the plunger wire was placed in aqueous samples for extraction. Choosing a suitable extracting solvent, some factors should be considered, such as good affinity with the etched wire to be stably held in the pores and the surface of the wire, good solubility for the analytes to ensure sufficiently high enrichment, low solubility in the aqueous solution, low vapor pressure to prevent potential loss during agitation, good chromatographic behaviar (the solvent peak must be well separated from the analyte peaks), low cost, ready availability, high purity and low toxicity. Based on these considerations above, from past experience and the solvent used in literature for LPME, o-xylene, n-hexane, propyl benzoate, toluene, and 1-octanol, were selected as potential solvents [45]. In order to compare the extraction efficiencies of these different organic solvents, based on past experience and parameters reported in other studies [25], solvent impregnation and extraction steps factors were set as follows: In the solvent impregnation process, the number of dynamic cycles was 5; 19 CHAPTER 3 the immersion time at each cycle was 1 min; and the agitation speed was set at 300 rpm (revolutions per minute). In the extraction process, flow rate was set at 0; agitation speed at 400 rpm; extraction time at 10 min. All parameters mentioned above would be optimised after the solvent selection step. The comparative results shown in Figure 3-1 indicated that 1-octanol gave higher extraction efficiency for all these five analytes compared with the other organic solvents and the bare etched wire. The bare etched wire shows the lowest extraction efficiency when compared to the wire loaded with organic solvent, which means the organic solvent played the dominant role in the extraction process. The poorer results obtained using other organic solvents might be due to a lower impregnation volume, which could be possibly due to a lower affinity for the etched wire surface or a lower solubility of the analytes in the organic solvent. It is possible that 1-octanol could be preferentially impregnated due to stronger interactions with the etched wire under this impregnation condition and the extraction parameters were favourable for extraction as far as 1-octanol was concened. As can be seen in Figure 3-1, different solvents showed different extraction efficiencies for the various analytes. As the target analytes were transferred from the aqueous sample to the organic phase, the extraction efficiency depended on the partition coefficients of the analytes between the aqueous and organic phases. As a result, different solvents showed different extraction efficiencies for different analytes. On the basis of the results, 1-octanol was chosen as the 20 CHAPTER 3 extraction solvent for the subsequent optimization. Figure 3-1 Comparison of extraction efficiency of five organic solvents and the etched wire without organic solvent for five organochlorine pesticides 3.2 Optimisation for solvent impregnation 3.2.1 Arrangement for preliminary optimisation The solvent impregnation technique that combines the dynamic cycle of the plunger wire movement in and out of the solvent and out and agitation was investigated. To evaluate and compare the performance of solvent impregnation under different conditions, 1-octanol was analyzed by thermal vaporation into the GC/MS system. Peak areas were measured and compared. There were three parameters (immersion time for each cycle, number of dynamic cycles and agitation speed) to be investigated. Table 3-1 shows the 21 CHAPTER 3 arrangement for preliminary optimisation. Table 3-1 Arragement for preliminary optimisation Number of Agitation speed Immersion time for dynamic cycle (rpm) each cycle (s) 1 1 0 30 2 1 0 60 3 1 350 30 4 1 350 60 5 1 700 30 6 1 700 60 7 5 0 30 8 5 0 60 9 5 350 30 10 5 350 60 11 5 700 30 12 5 700 60 Trial No. As shown in Table 3-1, there were 12 trials in the preliminary comparison of three parameters. The results of all 12 trials are shown in Figure 3-2. The white bar stands for immersion time of 30 seconds and black bar stands for that of 60 seconds. In Figure 3-2a, the number of dynamic cycles was 5, and in Figure 3-2b the number was 1. Each pair of column was at the same agitation speed, such as 0 rpm, 300 rpm and 600 rpm. 22 CHAPTER 3 Figure 3-2 Peak areas of 1-octanol under different agitation speeds, immersion time and the number of dynamic cycles As indicated in Figure 3-2, it is obvious that a 60-second immersion for each cycle performed better than 30-second immersion. So 1 min is the better choice for immersion time. The more times of dynamic cycles was applied, the higher 1-octanol signal was obtained. So more tests should be done to obtain the optimised number of cycles. The signal of 1-octanol of solvent impregnation process without agitation was the lowest in every group. However, the difference between signal intensity of 300 rpm and 600 rpm was not apparent. More settings of agitation speed need to be examined. 3.2.2 Optimisation of the number of dynamic cycles As described previously, the signal of 1-octanol was enhanced in relation to the increase of the number of dynamic cycles. One, 5 and 15 dynamic cycles were investigated at agitation speeds of 300 rpm and 600 rpm, 23 CHAPTER 3 respectively. Figure 3-3 shows the peak areas of 1-octanol under different numbers of dynamic cycles, and agitation speed. Figure 3-3 Influence of the number of dynamic cycles under different agitation speed (1, 5 and 15 in this figure stand for the number of dynamic cycles) In Figure 3-3, the white, gray and black bars in each group stand for 1, 5 and 15 times of dynamic cycles, respectively. The bars on the left were under the agitation speed of 300 rpm, and the ones on the right were under the agitation speed of 600 rpm. As can be seen in Figure 3-3, generally, the signals of 1-octanol in the 600-rpm agitation speed group were higher than those in 300-rpm agitation speed group. In the 600-rpm agitation speed group, the more the dynamic cycles were applied, the higher the 1-octanol signals, which was the same rule described above. However, the difference between 5 cycles and 15 cycles was not significant. In the 300-rpm agitation speed group the solvent peak intensity of 5 cycles was slightly higher than that of 15 cycles. Moreover, the peak intensity of 5 cycles of the 300-rpm group was higher than that of 15 cycles of the 600-rpm group. So 3, 5, 7 and 9 cycles under the 24 CHAPTER 3 agitation speed of 300 rpm were investigated afterwards. And the agitation speeds of 200 rpm, 300 rpm, 400 rpm and 500 rpm were investigated after the optimisation of the number of dynamic cycles was done. Figure 3-4 shows the comparison of the intensity of solvent signal under different times of dynamic cycles. Figure 3-4 Comparison of the intensity of solvent under diffrent numbers of dynamice times As shown in Figure 3-4, the peak intensity of 1-octanol increased as the number of cycles was raised, generally, except for the intensity of 3 cycles which was a little higher than that of 5 cycles. As shown in Figure 3-4, the optimised number of dynamic cycles was 9, under which condition the peak intensity was the highest and the RSD was the lowest among all four parameters. 25 CHAPTER 3 3.2.3 Optimisation of agitation speed As the number of dynamic cycles was determined, the optimisation of agitation speed process was carried out under the 9-dynamic-cycle condition. Figure 3-5 shows the signal intensity of 1-octanol under different agitation speeds. Figure 3-5 Signal intensity of 1-octanol under different agitation speed As can been seen in this figure, the intensity of the 1-octanol signal increased as the agitation speed was increased from 200 rpm to 400 rpm. However, when the agitation speed was raised to 500 rpm, the signal intensity of the solvent decreased. As the organic solvent held by the etched wire was exposed to the aqueous solution directly, it is conceivable that too high an agitation speed would lead to some solvent loss and possibly produce air bubbles that will influcence the impregnation effect, negatively. It is shown clearly in Figure 3-5 that the optimised agitation speed for 26 CHAPTER 3 solvent impregnation was 400 rpm. To calculate the volume of 1-octanol impregnated on the surface of the etched wire under the optimized impregnation conditions, a calibration curve ranging from 5 mg/g (methanol solvent) to 500 mg/g was developed. By substituting the peak area of the impregnation 1-octanol into the calibration curve, the volume of the 1-octanol was calculated to be 0.29 µL. 3.3 Extraction condition optimisation 3.3.1 Optimisation of agitation speed In other HPME techniques, such as HF-LPME [46], agitation of the sample solution could enhance the extraction process and reduce the time to achieve equilibrium. For HF-LPME, the organic solvent protected by the hollow fiber can tolerate higher stirring speeds. For SDME [2], in which the microdrop is directly exposed to the aqueous solution, higher agitation speed would result in loss of solvent, especially over a prolonged extraction time. In PIN-LPME, organic solvent was hold in the pores of the etched steel plunger wire, a situation which appear to be between those of HP-LPME and SDME. The agitation speed was investigated over the range from 300 and 600 rpm (since the higher limit of the agitator was 700 rpm) for 20 min. Figure 3-6 shows the extraction efficiencies for different analytes under different agitation speeds. As can be seen in Figure 3-6, extraction efficiencies of all five ananlytes 27 CHAPTER 3 were enhanced with an increase of the agitation speed from 300 rpm to 600 rpm. It is conceivable that too high a speed would lead to some solvent loss and possibly produce air bubbles that will affect repeatability and precision, but the result indicated that the high speed did not influence the extraction efficiencies and the opimised agitation speed for extraction in this study was 600 rpm. Figure 3-6 Influence of agitation speed on extraction effeciency 3.3.2 Optimisation of flow rate Continuous-flow microextracion is based on a flowing sample solution being in contact with the extraction phase directly [24]. There are two factors to be considered when selecting the optimum flow rate. One is stability of the organic solvent in continuous-flow microextraction; for example, in the SDME, the microdrop would face the possibility of being dislodged at high 28 CHAPTER 3 sample flow rates. The other factor is extracion dynamics. An increase in sample flow rate decreases the thickness of the interfacial layer surrounding the solvent surface, improving mass transfer of analytes, thus, speeding up the extraction. However, a reduction in peak areas is observed after exceeding a certain flow rate, which can be attributed to too high a linear velocity of the sample solution to allow establishment of an equilibrium in the interfacial layer of the two phases; the sample and the organic solvent[24]. Consequently, sample flow rates used in practice in continuous-flow microextraction ranges from 0.1-1.5 mL/min [25]. In this report, the flow rate was evaluated at 0, 0.2, 0.5 and 1 mL/min, respectively. The extraction efficiencies of different analytes under different flow rates are shown in Figure 3-7. As shown in Figure 3-7, when the flow rate was increased from 0 mL/min to 0.5 mL/min, the extraction efficiencies for all five analytes (except for Heptachlor and Dieldrin) increased accordingly. However, when the flow rate was increased from 0.5 mL/min to 1.0 mL/min, the efficiency of all five analytes decreased sharply. The phenomennon could be explained by the theory mentioned above that a high linear velocity of the sample solution may interfere with the establishment of extraction equilibrium between the sample and organic solvent [24]. High flow rates might also lead to the loss of extraction solvent from the etched wire, thus decreasing the extraction efficiency. 29 CHAPTER 3 It is clearly shown in Figure 3-7, that the optimum flow rate for the extraction of the five OCPs was 0.5 mL/min. Figure 3-7 Extraction efficiencies of different analytes under different flow rates (Agitation speed was 600 rpm, extraction time was 20 min) 3.3.3 Optimisation of extraction time The effect of extraction time between 5 and 40 min was investigated by extracting aqueous solutions containing 50 µg/L of each analyte at 600-rpm agitation speed, and the flow rate was set at 0.5 mL/min. The influence of extraction time on extraction efficiencies for five analytes is shown in Figure 3-8. The figure shows that the analytical signals increase quickly within 20 min of extraction time. Generally, in LPME, it is usually not practicable to prolong an extraction for equilibrium to be established. This is because the longer the extraction time, the greater the potential for solvent loss due to 30 CHAPTER 3 volatilization or dissolution in the sample solution. Additionally, it is more attractive to conduct time-efficient extraction process. As shown in Figure 3-8, the extraction efficiencies of 2,4'-DDT and 4,4'-DDT kept increasing as the expanding of the extraction time increased, while signals of the other three analytes decreased when the extraction time increased from 20 min to 40 min. Figure 3-8 Influence of extraction time on extraction efficiency As a consequence, taking the analysis time, solvent loss, good repeatability and precision and high extraction efficiency of this technique into consideration, an extraction time of 20 min was deemed to be the most favorable extraction time. 3.3.4 Desorption temperature and time optimisation Desorption temperature and time must be favorable to release all the analytes in the GC injector port. Four temperatures, 280°C, 285°C, 290°C, and 295°C, were examined to evaluate the effect of desorption temperature. The results are shown in Figure 3-9a. When desorption temperature was increased, 31 CHAPTER 3 analyte peak intensities were enhanced accordingly. For desorption time, 2 min, 5 min and 10 min, were examined to evaluate the effect on OCPs peak intensities. Figure 3-9b shows the results of comparison of desorption time. When desorption time was extented, analyte peak intensities were increased correspondingly. Figure 3-9 Signal Intensity of analytes under different desorption temperature (a) and desorption time (b). The overall results indicated that a temperature of 295°C was most favorable for complete desorption of all OCPs after 10 min. The performance of fully automated PIN-LPME is shown in Table 3-2, which includes the information on linear range, coefficients of determination (r2), limits of detection (LOD), relative standard deviations (RSD%) and 32 CHAPTER 3 enrichment factor (EFs) for all five OCPs. Table 3-2 Performance of fully-automated PIN- LPME Analyte Linear range (µg/L) r2 LOD (µg/L) RSD (%) (n=5) EF HCB 0.1-100 0.9954 0.01 2.8 611 Heptachlor 0.1-200 0.9965 0.02 11.9 208 Dieldrin 0.1-200 0.9991 0.02 6.7 108 2,4'-DDT 0.1-200 0.9991 0.02 9.9 465 4,4'-DDT 0.1-200 0.9996 0.02 11.5 878 Experiments were conducted under these optimized extraction conditions: in solvent impregnation step, 9 dynamic cycles, 400 rpm agitation speed; in extraction step, 0.5 mL/min flow rate, 600 rpm agitation speed, desorption temperature 295°C, desorption time 10 min. 3.4 Application to water samples Natural water from Macritchie Reservoir Singapore was collected and used as samples for evaluating the present fully automated PIN-LPME. None of the analytes were detected; either they were absent or their levels were below the LODs. Nevetheless, to evaluate the accuracy of the proposed method, relative recoveries were performed by spiking analyte standards into the reservoir water. The results of the analysis of these genuine water samples spiked at 10 µg/L of each analyte are shown in Table 3-4. As shown in Table 3-3, the relative recoveries ranged from 84% to 108%, which means that the matrix had no significant effect on PIN- LPME. The RSDs for them ranged from 5.4% to 11.1%. The results implied that the 33 CHAPTER 3 established method was reliable and applicable to real sample analysis. On the other hand the disadvantages of immersion SDME and SPME in terms of direct exposure to the sample matrix also apply to PIN-LPME, and it is anticipated that there would be significant matrix effects if very complex environmental sequeence samples were indentified. Table 3-3 Analysis of genuine water sample spiked at 10 µg/L of each analyte Analyte Relative recovery (%) RSD (%) (n=5) HCB 105 9.1 Heptachlor 84 11 Dieldrin 108 6.3 2,4'-DDT 96 10.6 4,4'-DDT 93 5.4 Figure 3-10 shows a GC/MS-SIM chromatogram of a spiked reservoir water sample after fully automated PIN-LPME. 2,4’-DDT HCB Dieldrin 4,4’-DDT Heptachlor Figure 3-10 GC/MS-SIM chromatogram of real samples after fully automated PIN-LPME: spiked reservoir water sample (spiked with 10 µg/L of each OCP). 34 CHAPTER 3 3.5 Comparison with other microextraction techniques LODs of the fully automated PIN-LPME for the OCPs were compared with those of other micoextraction techniques. The results are listed in Table 3-5. Table 3-4 LOD comparison of fully-automated PIN-LPME with other microextraction techniques (µg/L) Analyte Fully-automated PIN-LPME HF-LPME [47] DLLME [48] SDME [49] SPME [50] HCB 0.01 n.a. 0.0015 n.a. n.a. Heptachlor 0.02 0.030 0.01 0.049 0.00039 Dieldrin 0.02 0.047 0.0008 0.022 n.a. 2,4'-DDT 0.02 n.a. n.a. n.a. n.a. 4,4'-DDT 0.02 0.017 n.a. 0.101 0.00047 The LODs of the fully automated PIN-LPME for OCPs were comparable to those of HF-LPME and SDME techniques. Although DLLME and SPME gave slightly better LODs in some cases, the fully automated PIN-LPME was more convenient, of lower cost and as implemented in the present work, not only intepreted extrction and analysis automatically, but also allowed the automated sampling of water. The reason for the poorer performance of PIN-LPME could be the smaller volume and surface of extraction solvent comparing with DLLME and SPME. 35 CHAPTER 3 3.6 Conclusion A novel fully automated continuous-flow plunger-in-needle liquid phase microextraction (PIN-LPME) technique with gas chromatography/mass spectometric (GC/MS) analysis to determine five organochlorine pesticides (OCPs) from water samples was developed. A peristaltic pump was used to facilitate automated water sampling. With the utilisation of a CTC CombiPAL autosampler and its associated Cycle Composer software, a sample preparation-GC/MS method was feasible that allowed solvent impregnation, sample extraction, extract injection and analysis could be carried out completely automatically. The optimised conditions for solvent impregnation and extraction were as follows: 1-octanol as extraction solvent; impregnation mode of 9 times of dynamic cycles, 1 min of impregnation at 400 rpm for each cycle; extraction time of 20 min; agitation speed of 600 rpm; and sampling flow rate of 0.5 mL/min. Under the optimized conditions, good linearities for the OCPs were obtained. The limits of detection range from 0.01 to 0.02 µg/L. The enrichment factors ranged from 108 to 878, with relative standard deviations (RSDs) ranging from 2.8% to 11.9%. Finally, the developed method was succefully applied to the analysis of reservoir water sample. This automated continuous-flow PIN-LPME method demonstrated the feasibility of a complete analytical system comprising water sampling, sample preparation and GC/MS analysis that might be applied in on-site analysis of environmental samples, all automatically. 36 CHAPTER 3 3.7 Future work For the future, the developed technique can be used for the analysis of other types of analytes. Also, the automated system can be applied to complex matrices by possibly implementing an extra filtration setup. In our current study, the operation of the peristaltic pump for water sampling and CTC system for extraction and analysis were controlled separately, thus future efforts may be devoted to synchronize the pump to the CTC system. An integrated control system needs to be developed to facilitate communication between the two parts and further the scope of automation. If this is feasible, an integration of sampling, sampling preparation (extraction) and analysis of water for a fully automated platform may be realised. 37 REFERENCES [1] Jeannot, M. A. and Cantwell, F. F., Solvent microextraction into a single drop. Analytical Chemistry, 1996. 68(13): p. 2236-2240. [2] He, Y. and Lee, H. K., Liquid-phase microextraction in a single drop of organic solvent by using a conventional microsyringe. Analytical Chemistry, 1997. 69(22): p. 4634-4640. [3] Arthur, C. L. and Pawliszyn, J., Solid phase microextraction with thermal desorption using fused silica optical fibers. Analytical Chemistry, 1990. 62(19): p. 2145-2148. [4] Pawliszyn and Janusz, Handbook of solid phase microextraction. Oxford; Chennai: Elsevier, 2011. [5] Gorlo, D., Zygmunt, B., Dudek, M., Jaszek, A., Pilarczyk, M., and Namieśnik, J., Application of solid-phase microextraction to monitoring indoor air quality. Fresenius' Journal of Analytical Chemistry, 1999. 363(7): p. 696-699. [6] Wei, L., Ou, Q., Li, J., and Liang, B., Preparation of a solid-phase microextraction fiber coated with γ-Al2O3 and determination of volatile organic compounds in indoor air. Chromatographia, 2004. 59(9): p. 601-605. [7] Larroque, V., Desauziers, V., and Mocho, P., Development of a solid phase microextraction (SPME) method for the sampling of VOC traces in 38 indoor air. Journal of Environmental Monitoring, 2006. 8(1): p. 106-111. [8] Khaled, A. and Pawliszyn, J., Time-weighted average sampling of volatile and semi-volatile airborne organic compounds by the solid-phase microextraction device. Journal of Chromatography A, 2000. 892(1): p. 455-467. [9] Müller, L., Górecki, T., and Pawliszyn, J., Optimization of the SPME device design for field applications. Fresenius' Journal of Analytical Chemistry, 1999. 364(7): p. 610-616. [10] Xiong, G., Goodridge, C., Wang, L., Chen, Y., and Pawliszyn, J., Microwave-assisted headspace solid-phase microextraction for the analysis of bioemissions from Eucalyptus citriodora leaves. Journal of Agricultural and Food Chemistry, 2003. 51(27): p. 7841-7847. [11] Xiong, G., Chen, Y., and Pawliszyn, J., On-site calibration method based on stepwise solid-phase microextraction. Journal of Chromatography A, 2003. 999(1): p. 43-50. [12] da Costa Silva, R. G. and Augusto, F., Highly porous solid-phase microextraction fiber coating based on poly (ethylene glycol)-modified ormosils synthesized by sol–gel technology. Journal of Chromatography A, 2005. 1072(1): p. 7-12. [13] Zuin, V. G., Lopes, A. L., Yariwake, J. H., and Augusto, F., Application of a novel sol–gel polydimethylsiloxane–poly (vinyl alcohol) solid-phase microextraction fiber for gas chromatographic determination of pesticide 39 residues in herbal infusions. Journal of Chromatography A, 2004. 1056(1): p. 21-26. [14] Lopes, A. L. and Augusto, F., Preparation and characterization of polydimethylsiloxane/poly microextraction fibers (vinylalcohol) using sol–gel coated technology. solid phase Journal of Chromatography A, 2004. 1056(1): p. 13-19. [15] Azenha, M., Malheiro, C., and Silva, A. F., Ultrathin phenyl-functionalized solid phase microextraction fiber coating developed by sol–gel deposition. Journal of Chromatography A, 2005. 1069(2): p. 163-172. [16] Giardina, M. and Olesik, S. V., Application of low-temperature glassy carbon films in solid-phase microextraction. Analytical Chemistry, 2001. 73(24): p. 5841-5851. [17] Giardina, M., Ding, L., and Olesik, S. V., Development of fluorinated low temperature glassy carbon films for solid-phase microextraction. Journal of Chromatography A, 2004. 1060(1): p. 215-224. [18] Dong, C., Zeng, Z., and Li, X., Determination of organochlorine pesticides and their metabolites in radish after headspace solid-phase microextraction using calix [4] arene fiber. Talanta, 2005. 66(3): p. 721-727. [19] Zeng, Z., Qiu, W., and Huang, Z., Solid-phase microextraction using fused-silica fibers coated with sol-gel-derived hydroxy-crown ether. 40 Analytical Chemistry, 2001. 73(11): p. 2429-2436. [20] Wang, D., Xing, J., Peng, J., and Wu, C., Novel benzo-15-crown-5 sol– gel coating for solid-phase microextraction. Journal of Chromatography A, 2003. 1005(1): p. 1-12. [21] Kokosa, J. M., Advances in solvent-microextraction techniques. TrAC Trends in Analytical Chemistry, 2013. 43(1): p. 2-13. [22] Liu, S. and Dasgupta, P. K., Liquid droplet a renewable gas sampling interface. Analytical Chemistry, 1995. 67(13): p. 2042-2049. [23] Ouyang, G., Zhao, W., and Pawliszyn, J., Automation and optimization of liquid-phase microextraction by gas chromatography. Journal of Chromatography A, 2007. 1138(2): p. 47-54. [24] Liu, W. and exceeding1000-fold Lee, H. K., concentration Continuous-flow of dilute microextraction analytes. Analytical Chemistry, 2000. 72(18): p. 4462-4467. [25] Kokosa, J. M., Przyjazny, A., and Jeannot, M., Solvent microextraction: theory and practice: John Wiley & Sons, 2009. [26] Psillakis, E. and Kalogerakis, N., Application of solvent microextraction to the analysis of nitroaromatic explosives in water samples. Journal of Chromatography A, 2001. 907(1): p. 211-219. [27] Xu, L., Basheer, C., and Lee, H. K., Developments in single-drop microextraction. Journal of Chromatography A, 2007. 1152(1): p. 184-92. 41 [28] Pedersen-Bjergaard, S. and Rasmussen, K. E., Liquid-liquid-liquid microextraction for sample preparation of biological fluids prior to capillary electrophoresis. Analytical Chemistry, 1999. 71(14): p. 2650-2656. [29] Guo, L. and Lee, H. K., Electro membrane extraction followed by low-density solvent microextraction based combined ultrasound-assisted with derivatization emulsification for determining chlorophenols and analysis by gas chromatography–mass spectrometry. Journal of Chromatography A, 2012. 1243(1): p. 14-22. [30] Bedendo, G. C., Jardim, I. C. S. F., and Carasek, E., Multiresidue determination of pesticides in industrial and fresh orange juice by hollow fiber microporous membrane liquid–liquid extraction and detection by liquid chromatography–electrospray-tandem mass spectrometry. Talanta, 2012. 88(1): p. 573-580. [31] Bedendo, G. C., Jardim, I. C. S. F., and Carasek, E., A simple hollow fiber renewal liquid membrane extraction method for analysis of sulfonamides in honey samples with determination by liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 2010. 1217(42): p. 6449-6454. [32] Liu, W., Wei, Z., Zhang, Q., Wu, F., Lin, Z., Lu, Q., Lin, F., Chen, G., and Zhang, L., Novel multifunctional acceptor phase additive of water-miscible ionic liquid in hollow-fiber protected liquid phase 42 microextraction. Talanta, 2012. 88(1): p. 43-49. [33] Ghambarian, M., Yamini, Y., and Esrafili, A., Developments in hollow fiber based liquid-phase microextraction: principles and applications. Microchimica Acta, 2012. 177(3): p. 271-294. [34] Guo, L. and Lee, H. K., Ionic liquid based three-phase liquid–liquid– liquid solvent bar microextraction for the determination of phenols in seawater samples. Journal of Chromatography A, 2011. 1218(28): p. 4299-4306. [35] Payán, M. R., López, M. Á. B., Torres, R. F., Navarro, M. V., and Mochón, M. C., Electromembrane extraction (EME) and HPLC determination of non-steroidal anti-inflammatory drugs (NSAIDs) in wastewater samples. Talanta, 2011. 85(1): p. 394-399. [36] Lee, J., Lee, H. K., Rasmussen, K. E., and Pedersen-Bjergaard, S., Environmental and bioanalytical applications of hollow fiber membrane liquid-phase microextraction: a review. Analytica Chimica Acta, 2008. 624(2): p. 253-68. [37] Breadmore, M. C., Ionic liquid-based liquid phase microextraction with direct injection for capillary electrophoresis. Journal of Chromatography A, 2011. 1218(10): p. 1347-1352. [38] Yan, H., Wang, H., Qiao, J., and Yang, G., Molecularly imprinted matrix solid-phase dispersion combined with dispersive liquid–liquid microextraction for the determination of four Sudan dyes in egg yolk. 43 Journal of Chromatography A, 2011. 1218(16): p. 2182-2188. [39] Rezaee, M., Yamini, Y., Moradi, M., Saleh, A., Faraji, M., and Naeeni, M. H., Supercritical fluid extraction combined with dispersive liquid– liquid microextraction as a sensitive and efficient sample preparation method for determination of organic compounds in solid samples. The Journal of Supercritical Fluids, 2010. 55(1): p. 161-168. [40] Wang, H., Li, G., Zhang, Y., Chen, H., Zhao, Q., Song, W., Xu, Y., Jin, H., and Ding, L., Determination of triazine herbicides in cereals using dynamic microwave-assisted extraction with solidification of floating organic drop followed by high-performance liquid chromatography. Journal of Chromatography A, 2012. 1233(1): p. 36-43. [41] Ranjbari, E., Biparva, P., and Hadjmohammadi, M. R., Utilization of inverted dispersive liquid–liquid microextraction followed by HPLC-UV as a sensitive and efficient method for the extraction and determination of quercetin in honey and biological samples. Talanta, 2012. 89(1): p. 117-123. [42] Zhang, H., Ng, B. W., and Lee, H. K., Development and evaluation of plunger-in-needle liquid-phase microextraction. Journal of Chromatography A, 2014. 1326(1): p. 20-8. [43] Xu, H., Li, Y., Jiang, D., and Yan, X., Hydrofluoric acid etched stainless steel wire for solid-phase microextraction. Analytical Chemistry, 2009. 81(12): p. 4971-4977. 44 [44] Zuccher, S., Experimental investigations of the liquid-film instabilities forming on a wire under the action of a die. International Journal of Heat and Fluid Flow, 2008. 29(6): p. 1586-1592. [45] Lee, J. and Lee, H. K., Fully automated dynamic in-syringe liquid-phase microextraction and on-column derivatization of carbamate pesticides with gas chromatography/mass spectrometric analysis. Analytical Chemistry, 2011. 83(17): p. 6856-6861. [46] Shen, G. and Lee, H. K., Hollow fiber-protected liquid-phase microextraction of triazine herbicides. Analytical Chemistry, 2001. 74(3): p. 648-654. [47] Basheer, C., Balasubramanian, R., and Lee, H. K., Determination of organic micropollutants membrane/liquid-phase in rainwater microextraction using combined hollow fiber with gas chromatography–mass spectrometry. Journal of Chromatography A, 2003. 1016(1): p. 11-20. [48] Zhang, Y. and Lee, H. K., Application of ultrasound-assisted emulsification microextraction based on applying low-density organic solvent for the determination of organochlorine pesticides in water samples. Journal of Chromatography A, 2012. 1252(1): p. 67-73. [49] Cortada, C., Vidal, L., Tejada, S., Romo, A., and Canals, A., Determination of organochlorine pesticides in complex matrices by single-drop microextraction coupled to gas chromatography–mass 45 spectrometry. Analytica Chimica Acta, 2009. 638(1): p. 29-35. [50] Banitaba, M. H., Mohammadi, A. A., Hosseiny Davarani, S. S., and Mehdinia, A., Preparation and evaluation of a novel solid-phase microextraction fiber based on poly(3,4-ethylenedioxythiophene) for the analysis of OCPs in water. Analytical Methods, 2011. 3(9): p. 2061-2067. 46 [...]... syringe and analyzed by GC As reported, DLLME has many applications in sample analysis, such as clozapin in unrine and serum [37], Sudan dyes in egg yolk [38], PAHs in marine sediments [39], hebicides in cereals [40], quercetin in honey [41], etc 6 CHAPTER 1 1.1.3 Plunger- in- needle liquid- phase microextraction Recently, a novel LPME technique called plunger- in- needle liquid- phase microextraction (PIN-LPME)... consumption was much reduced [42] Therefore, in this report, 1 A fully automated continuous-flow plunger- in- needle liquid- phase microxtraction (PIN-LPME) system is reported; 2 Several organochlorine pesticides are selected as model analytes to evaluate the procedure; 3 Parameters influencing the impregnation of extractant and the performance of PIN-LPME are investigated and optimized; 4 This system is... the drop back into the syringe needle and then pushing it back out repeatedly, which was called in- needle dynamic modes LPME [23], and 3) extracting analytes from a continuous flow of sample solution [24] SDME can be fully automated using a computer-programmable autosampler, such as a CTC CombiPAL using patented software [25] However, in practical applications, forces generated by stirring of the aqueous... the PIN-LPME device is illustrated in Figure 1-4 Figure 1-4 Schematic of the home-assembled PIN-LPME device The stainless steel plunger wire of a commercial plunger- in- needle microsyringe was etched with hydrofluoric acid to form a microporous structure, and the etched plunger was used as the extractant solvent holder Figure 1-5 shows the scanning electron micrographs of the surface of the stainless... Spectra-Teknik, Singapore The plunger- in- needle syringe with replaceable 26-gauge, 70 mm long needle, 0.47mm internal diameter (I.D.) microsyringe (0.5-µL capacity) was purchased from SGE (Ringwood, VIC, Australia) For LPME applications, a replacement needle (23-gauge, 50 mm long needle, 0.63 mm I.D., SGE) was necessary The latter one with wider bore and shorter needle allowed the plunger, particularly the... injection port of the GC for 30 min 2.4.2 Solvent impregnation In the solvent impregnation step, the etched part was immersed in organic solvent to allow full impregnation of the solvent into the pores The whole process is presented as follow: The plunger wire was pulled back into the needle Then the needle was inserted into the solvent vial, which is placed in the agitator The plunger was pushed out to make... extraction process Figure 2-3 Schematic of the PIN-LPME device in extraction step As shown in Figure 2-3, the continuous flow was formed by pumping the water sample from the reservoir into and out of the sample vial during the extraction process After extraction for a certain time under a certain agitation speed, the plunger wire was withdrawn into the needle for protection, and the assembly was removed... stainless steel wire before and after etching The extractant could be more easily held within the pores, comparing with the drop in the tip of needle in SDME When the plunger wire with the extractant was exposed to the sample solution, analytes diffused from the sample solution to the extractant After 7 CHAPTER 1 extraction, the plunger wire was directly introduced into the injection port of a GC/MS system... out to make the etched wire immersed into the extraction solvent for a certain period of time, which need to be optimised During the immersion period, the agitator was shaking at a certain speed After the certain period of time, the agitator was stopped and the plunger was pulled back into the needle The cycle of steps from the immersion to the pulling back of the plunger were repeated for several times,... The etching steps were as follows: the plunger wire was cleaned in acetone, wiped with a piece of lint-free tissue and immersed in hydrofluoric acid for 15 min at room temperature The etched part of the plunger wire was 2 cm long After etching, the plunger wire was washed gently with ultrapure water and dried under room temperature for 1 13 CHAPTER 2 hour and then conditioned at 300°C in the injection ... hebicides in cereals [40], quercetin in honey [41], etc CHAPTER 1.1.3 Plunger- in- needle liquid- phase microextraction Recently, a novel LPME technique called plunger- in- needle liquid- phase microextraction. .. novel fully automated continuous-flow plunger- in- needle liquid phase microextraction (PIN-LPME) technique with gas chromatography/mass spectometric (GC/MS) analysis to determine five organochlorine... analytes into one device and organic solvent consumption was much reduced [42] Therefore, in this report, A fully automated continuous-flow plunger- in- needle liquid- phase microxtraction (PIN-LPME)