Fully Automated Headspace Bubble-in-Drop Microextraction

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Fully Automated Headspace Bubble-in-Drop Microextraction Liang Guo, Nurliyana Binte Nawi, and Hian Kee Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01543 • Publication Date (Web): 29 Jul 2016 Downloaded from http://pubs.acs.org on August 1, 2016

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Analytical Chemistry

Fully Automated Headspace Bubble-in-Drop Microextraction Liang Guo,a,b Nurliyana binte Nawi,a Hian Kee Lee*,a,b a

Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore

117543, Singapore b

National University of Singapore Environmental Research Institute, T-Lab Building #02-01,

5A Engineering Drive 1, Singapore 117411, Singapore

*Corresponding author. Tel.: +65 6516 2995; fax: +65 6779 1691

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ABSTRACT A fully-automated headspace bubble-in-drop microextraction (automated HSBID) method, coupled to gas chromatography/mass spectrometric (GC/MS) analysis, was developed for the analysis of nitro musks in environmental water samples. The entire procedure, including the extraction of the analytes by HS-BID and GC/MS analysis of the analyte-enriched solvent, was completely automated. In BID, a certain volume of air is introduced into the extraction solvent droplet, enlarging the surface area of the extraction solvent droplet in relation to the water sample without increasing its volume, significantly enhancing extraction efficiency. Compared to conventional single drop microextraction, the developed method has higher extraction efficiency due to the enlarged surface area of the extraction solvent droplet. Under the optimized conditions (1.0 mL of sample solution, using 1.0 µL of 1-octanol containing of 0.5 µL of air bubble, at 40 ℃ for extraction for 20 min), the automated HS-BID gave low limits of detections (between 0.012 and 0.042 µg/L), good linearity (from 0.1 to 20 µg/L, and from 0.2 to 50 µg/L, with r2 between 0.9909 and 0.9958, depending on analytes), and good repeatability of the extractions (relative standard deviations, below 4.7%, n=5). The developed procedure was applied to determine nitro musks in environmental water samples, and was demonstrated to be efficient, labour-free, economical, and environmentally benign.


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INTRODUCTION Liquid-phase microextraction (LPME), as a microscale variation of liquid-liquid extraction (LLE), was introduced in the mid-to-late 1990s.1,2 In LPME, analytes are extracted from an aqueous sample solution into a water-immiscible extraction solvent. In the original LPME methodology, since only a small volume of organic solvent, usually in the microlitre range, was used, the solvent was in the form of a drop, this specific form of LPME was named single-drop microextraction (SDME).3 To perform SDME, a droplet of the extraction solvent is suspended at the tip of the needle of a microsyringe and immersed in the aqueous sample solution for extraction, thus, this mode of SDME is termed direct immersion (DI)-SDME. After extraction, the solvent droplet is withdrawn back into the microsyringe, and then injected into an instrument for analysis. Characterized by high efficiency, and simplicity of use and operation, SDME has been widely used for the analysis of various analytes. 4-15 In general, a higher stirring speed facilitates the mass transfer of analyte from sample solution into the extraction solvent droplet, enhancing the extraction efficiency. However, in DI-SDME a higher stirring rate may lead to the instability of the solvent droplet and possibly detaching it from the tip of the needle. In addition, the extraction solvent droplet may also be unstable when immersed in a complex sample solution. Theis et al

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introduced a new mode of SDME, named headspace (HS)-SDME, in which

the extraction solvent droplet is suspended in the headspace of an aqueous sample solution and is not contact with the sample solution directly. This means that in HS-SDME, matrix effects are reduced. Analytes are distributed from the aqueous sample solution into the headspace, and then extracted into the solvent droplet. The aqueous sample solution may be stirred at a higher speed to facilitate the mass transfer, accelerating the extraction in comparison with DI-SDME. Since the analytes are distributed from the sample solution into the headspace, HS-SDME is more suitable for the extraction of volatile analytes. Avoiding, or greatly reducing, the matrix effects of the sample solution on the extraction solvent droplet, HS-SDME can be applied to more complex sample matrices. Since its introduction, HSSDME has seen application in different fields. 17-25 Recently, an alternative of SDME, termed as bubble in drop single-drop microextraction (BID-SDME), has been reported.26 In this method, a droplet of extraction solvent is suspended at the tip of the microsyringe, and a certain volume of air bubble is introduced into the droplet during the extraction procedure. Thus, the intentional addition of an air bubble of defined volume improved the extraction efficiency due to an increase of the surface area of ACS Paragon3Plus Environment


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the droplet and result in a thin film effect that enhanced mass transfer.26 The bubble in drop method has been applied to extract atrazine and metolachlor

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and other pesticides,28 and

growth homones. 29 However, there are some shortcomings of the BID-SDME approach. Firstly, because the extraction solvent droplet is directly immersed in the aqueous sample solution, it would be potentially unstable and could be easily detached from the needle tip, especially under a high extraction temperature or a high stirring rate. Secondly, the procedure hitherto has been performed manually, thus limiting its applicability for extracting a large number of samples, the operations being tedious and labour-intensive. To address the current shortcomings of conventional BID-SDME, a novel approach of this procedure, termed automated headspace bubble-in-drop microextraction (HS-BID), coupled to gas chromatography/mass spectrometric (GC/MS) analysis, was developed. All steps of the procedure including the expulsion of the solvent droplet from the microsyringe to the tip of the needle, introduction of the air bubble into the droplet, withdrawal of the solvent droplet back into the microsyringe after extraction, and the injection of the analyte-enriched droplet into a GC/MS system for analysis, was carried out automatically. Moreover, the headspace mode obviates problems caused by complex matrices. Parameters affecting the extraction efficiency (the type of extraction solvent, the volume of air bubble, extraction temperature, extraction time, and effect of salt addition) were evaluated. The optimized conditions were applied to the analysis of musks from genuine water samples to demonstrate the feasibility of automated HS-BID-GC/MS.

EXPERIMENTAL SECTION Chemical and Materials. Musk ambrette, musk xylene, musk moskene, and musk tibeten standards were bought from Dr. Ehrenstorfen GmbH (Augsburg, Germany), while musk ketone was bought from SAFC, Sigma (St. Louis, MO, 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. Automated Headspace BID. The entire automated HS-BID procedure including analyte extraction by BID and introduction of the final extract into the GC/MS system was carried out on a CTC Analytics autosampler (Zwingen, Switzerland) (coupled to a GC/MS system) with the aid of the Cycle Composer software (CTC Analytics). ACS Paragon4Plus Environment

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The optimized automated HS-BID procedure was conducted as follows (Figure 1): 1.0 mL sample solution was placed in a sample vial, in which sodium chloride was added, to give a concentration of 10% (w/v). Air (0.5 µL) was withdrawn into the microsyringe from an empty sample vial, followed by withdrawing 1.0 µL of 1-octanol from the extraction solvent vial. The microsyringe was then moved to the sample vial, and its needle was inserted into the headspace after piercing the vial septum and was suspended over the sample solution in a fixed position. The plunger was depressed such that the extraction solvent was expelled from the microsyringe and was suspended at the tip of the microsyringe needle. Then 0.5 µL of air was introduced into the droplet of the extraction solvent. The extraction solvent droplet with air bubble inside was exposed to the headspace above the aqueous sample solution for extraction. After 20 min of extraction, the extraction solvent droplet was retracted into the microsyringe. The microsyringe moved from the sample vial, and the extract is introduced into the GC/MS system for analysis. The entire HS-BID procedure was automatically repeated to extract and analyze the subsequent samples sequentially. Automated Headspace SDME. Automated HS-SDME was performed in the same way as that of automated HS-BID, except no air bubble was introduced into the extraction solvent droplet for the extraction. GC/MS Analysis. Details of GC/MS analysis are provided as Supporting Information.

RESULTS AND DISCUSSION In order to evaluate the automated HS-BID various parameters affecting the extraction efficiency, including the type of extraction solvent, the volume of air bubble, extraction time and temperature, and effect of salt addition, were studied. Selection of Extraction Solvent. As in other modes of LPME, for HS-BID, the organic solvent was a critical parameter on the extraction. First of all, the extraction solvent should have high extraction capability for the target analytes. Second, the extraction solvent should have a relatively lower vapor pressure to avoid evaporation of the solvent droplet during the extraction process. Third, for this proposed method, the solvents should be stable enough to remain at the tip of the microsyringe needle throughout the extraction. Four organic solvents including 1-octanol, toluene, hexane, and chloroform were evaluated as extraction solvents. The higher peak areas were obtained using 1-octanol when compared with the other solvents (shown in Figure S-1). For the developed approach, a solvent with a lower vapor pressure will undergo relatively lower losses during extraction, which would be partly beneficial to the extraction. For the four solvents used here, 1-octanol has the lowest vapor pressure at 0.14 ACS Paragon5Plus Environment


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mmHg (25 °C) compared to chloroform at 160 mmHg (25 °C), toluene at 26 mmHg (25 °C) and n-hexane at 132 mmHg (20 °C). The lower vapor pressure of 1-octanol thus contributed relatively positively to the extraction. This observation was in line with a previous report.30 Air Bubble Volume. To evaluate the effect of air bubble volume on extraction, different volumes (0.2, 0.4, 0.5, 0.6, and 0.8 µL) were studied with 1.0 µL of extraction solvent. The results are illustrated in Figure 2, which shows an obvious increase in the peak areas of all analytes with the increase of the volume of the air bubble from 0.2 µL to 0.5 µL. With further increase of the volume from 0.5 µL to 0.8 µL, no significant increases in peak areas of the analytes was observed. It was noted that when the air bubble size was bigger than 0.5 µL, the extraction solvent droplet would be unstable, being easily detached from the needle tip. A 1.0 µL of extraction solvent droplet with 0.5 µL of air bubble inside remained stable at the tip of the microsyringe during the extraction procedure; no detachment of extraction solvent droplets of this volume was experienced in any of our experiments. Thus, a volume of 0.5 µL of air bubble was adopted for subsequent experiments. Extraction Time Profile. A series of experiments was conducted to evaluate the effect of extraction times (5, 10, 15, 20, and 30 min). The extraction time profiles are shown in Figure S-2. The peak areas of all analytes increased as the extraction time increased from 5 to 20 min. After then, the peak areas for most analytes reached a plateau, except that the peak areas for ambrette and xylene decreased slightly. Over a prolonged extraction time, evaporation of the extraction solvent droplet is possible, affecting the extraction negatively. On the basis of this observation, a 20-min extraction time was deemed to be most favorable. Extraction Temperature. In HS-BID, the mass transfer of analyte from the aqueous sample solution into the headspace and then from the headspace into the extraction solvent droplet was temperature-dependent. A series of extractions was conducted under different temperature conditions in the range of 30 °C to 60 °C with 10 °C increments, as well as at 25 °C (ambient temperature), to investigate their effects on extraction. The results are shown in Figure S-3. The peak areas for all analytes increased significantly with the increase of temperature of up to 40 °C. The peak areas then increased slowly or reached a plateau. Considering the balance of high extraction efficiency and to avoid the possibility of loss of solvent via evaporation at relatively higher temperatures, 40 °C was chosen as the most favorable temperature. Salt Effect. The salting-in-effect has been widely used in extractions from water, including LPME and solid-phase microextraction (SPME), to decrease the solubility of


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analytes in the aqueous sample solution, thus increasing their partition into the extraction solvent,31 or in the present case, the headspace.32,33 Various amounts of sodium chloride (ranging from 0 to 30%, w/v) were added to the sample solution to evaluate the influence of salt addition on extraction, The results (shown in Figure S-4) indicated that the highest peak areas for most analytes were achieved when 10% of salt was added. With more than 10% salt, no significant increases in peak areas were observed. Comparison with conventional SDME. For HS-BID, due to the introduction of a certain volume of air bubble in the extraction solvent droplet, the surface area of the solvent droplet would be greatly enlarged, leading to more efficient extraction. The HS-BID approach (using 1.0 µL of extraction solvent containing 0.5 µL of air bubble inside) was compared to conventional HS-SDME with different volumes (1.0 µL and 1.5 µL) of extraction solvent. From Figure 3, it can be seen that the peak areas for all analytes obtained by HS-BID were much higher than those obtained by HS-SDME using 1.0 µL of extraction solvent, and even higher than those obtained by HS-SDME using 1.5 µL of extraction solvent. The results indicated that the encapsulated air bubble in the extraction solvent droplet was a contribution factor to the enhancement of extraction efficiency, enhancing the surface area of the solvent droplet without increasing its volume (which would compromise the enrichment factor). Method Evaluation. Under the most favorable conditions, the procedure was validated by studying the linearity range, limits of detection (LODs), limits of quantification (LOQs), repeatability, and recoveries by extracting spiked ultrapure water samples. The results are shown in Table 1. Good linearity of the calibration plots based on analyte peak areas was obtained from 0.1 to 20 µg/L (musk moskene, musk tibeten, and musk ketone), and from 0.2 to 20 µg/L (musk ambrette and musk xylene). The regression coefficients for all analytes were higher than 0.9909. The repeatability of the method was evaluated by five replicate analyses of the spiked water samples under the same operational parameters, and presented as the relative standard deviations (RSDs), which were between 3.1% and 4.7%, showing the good repeatability of the method. The LODs, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.012 to 0.042 µg/L. The LOQs, based on an S/N ration of 10, ranged from 0.041 to 0.141 µg/L. The results obtained are comparable or better than several other extraction methods like ionic liquid based

HS-SDME-GC/MS/MS,24

dispersive liquid-liquid


microextraction

(DLLME)-


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GC/MS,34,35 solvent demulsification DLLME-liquid chromatography/MS,36 packed sorbents microextraction-GC/MS,37 dispersive micro-solid phase extraction-GC/MS,38 microwaveassisted HS-SPME-GC/MS,39 ultrasound-assisted emulsification microextraction.40 The current method is fully automated, integrating headspace BID and GC/MS analysis seamlessly. All steps of the method could be automatically conducted in consecutive multiple extractions. Additionally, the current method demonstrated a particular advantage in the total amount of organic solvent consumed (only several microliters). Analysis of Genuine Water Samplers. The automated HS-BID-GC/MS was applied to analyze genuine and spiked river water samples, to evaluate its applicability. No analytes were found in the genuine samples, indicating they were free of the target analytes or the concentrations of the target analytes were below the LODs of the procedure. The river water samples were then spiked with musk standards at concentrations of 0.5 and 5 µg/L of each analyte and extracted with the developed method to evaluate matrix effects. The relative recoveries, used to evaluate matrix effects and defined as the ratios of the peak areas of the analytes in river water extracts to peak areas of the analytes in ultrapure water extracts spiked at the same concentrations, were calculated, and are listed in Table 2. The relative recoveries were between 92.1% and 104.5%, with RSDs (n=5) of

Fully Automated Headspace Bubble-in-Drop Microextraction

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