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Capillary electrophoretic determination of ammonia using headspace single-drop microextraction
Microchemical Journal 86 (2007) 48 – 52 www.elsevier.com/locate/microc
Capillary electrophoretic determination of ammonia using headspace single-drop microextraction Birutė Pranaitytė a , Svetlana Jermak a , Evaldas Naujalis b , Audrius Padarauskas a,⁎ a
Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania Laboratory for Metrology in Chemistry, Semiconductor Physics Institute, A. Gostauto 11, LT-01108 Vilnius, Lithuania
b
Received 28 August 2006; received in revised form 10 October 2006; accepted 10 October 2006 Available online 17 November 2006
Abstract A new method involving headspace single-drop microextraction (SDME) and capillary electrophoresis (CE) is developed for the preconcentration and determination of ammonia (as dissolved NH3 and ammonium ion). An aqueous microdrop (5 μL) containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 (as internal standard) was used as the acceptor phase. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time) affecting the extraction efficiency were investigated. Proposed SDME-CE method provided about 14-fold enrichment in about 20 min. The calibration curve was linear for concentrations of NH+4 in the range from 5 to 100 μmol/L (R2 = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/L of NH+4 . Such detection sensitivity is high enough for ammonia determination in common environmental and biological samples. Finally, headspace SDME was applied to determine ammonia in human blood, seawater and milk samples with spiked recoveries in the range of 96–107%. © 2006 Elsevier B.V. All rights reserved. Keywords: Single-drop microextraction; Capillary electrophoresis; Ammonia
1. Introduction Ammonia is a widely occurring natural compound, often produced as a consequence of microbial protein catabolism, and thus serves as a quality indicator of fruit juice, milk, cheese, meat, seafood, and bakery products. In the wine industry, ammonia determination is important in the calculation of yeast available nitrogen. It is also monitored in swimming pool water, waste water, and fermentation cultures, where its detection may indicate the presence of faeces, urine and microbes. An elevated ammonia blood level is considered a strong indicator of an abnormality in nitrogen homeostasis, the most common related to liver dysfunction. Ammonia is also an important micronutrient and intermediate of the nitrogen cycle in aquatic ecosystem. High concentrations of ammonia in water can lead to nutrient enrichment of water systems, and the subsequent increase in biological activity can lead to algal blooms the potential for turbidity, taste, odor and toxicity problems. There⁎ Corresponding author. Fax: +370 5 2330987. E-mail address: [email protected] (A. Padarauskas). 0026-265X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2006.10.001
fore, the development of a rapid and simple analytical technique for the monitoring of ammonia (as dissolved NH3 and ammonium ion) in various samples is of significant interest. There are many methods used for the determination of ammonia or ammonium ion [1–4]. However, the determination of ammonia in the samples with complicated matrices requires the separation of ammonia from sample matrix which is usually achieved through alkaline distillation [5], aeration/microdiffusion [6] or solid-phase extraction [7]. Each method has its own merits, but they are laborious, time-consuming and often lack sensitivity. In the past decade, one noticeable trend in the development of sample preparation techniques has been miniaturization [8]. Miniaturized modification of the traditional liquid–liquid extraction method termed single-drop microextraction (SDME) is one of the recent trends in this area [9–11]. In practice, two main approaches can be used to perform single-drop microextractions. In direct single-drop microextraction, a drop of a water-immiscible solvent is suspended directly from the tip of a microsyringe needle immersed in the aqueous sample. This extraction approach, in combination with GC or
B. Pranaitytė et al. / Microchemical Journal 86 (2007) 48–52
HPLC, has been shown to be quite effective for extraction and analysis of various organic compounds [12]. However, applications of direct SDME are normally restricted to medium polarity and non-polar analytes and those whose polarities can be reduced before extraction. Furthermore, the organic solvents used for direct SDME which are immiscible with water, are not compatible with conventional capillary electrophoresis (CE). An alternative way of minimizing some of these problems is to use headspace SDME. In this approach a microdrop of appropriate solvent is placed in the headspace of the sample solution [13–15] or in a flowing air sample stream [9,16–18] to extract volatile analytes. Headspace SDME has similar capabilities in terms of precision and speed of analysis compared to direct SDME but has an advantage to choice of a wider variety of solvents. Unlike direct SDME, water can be also used as the solvent to extract volatile and water soluble analytes. This significantly enhances the range extractable analytes as well as the range of analytical methods that can be coupled with SDME. In addition, headspace SDME was found to provide excellent clean-up from the samples with complicated matrices [19,20]. The aim of this work was to provide a new approach for the rapid and simple monitoring of ammonia involving water-based headspace single-drop microextraction with subsequent capillary electrophoretic determination. The feasibility of the method was demonstrated by the analysis of human blood, milk and seawater samples. 2. Experimental 2.1. Instrumentation Separations were performed on a P/ACE 2100 apparatus (Beckman Instruments Inc., Fullerton, CA, USA) equipped with a UV detector with wavelength filters (200, 214, 230 and 254 nm). Fused silica capillaries (Polymicro Technology, Phoenix, AZ, USA) of 75 μm i.d. and 57 cm total length (50 cm to the detector) were used. Samples were injected in the hydrodynamic mode by overpressure (3.43 × 103 Pa). System Gold software (Beckman Instruments Inc.) was used for data acquisition. Indirect UV detection (214 nm) was employed at the cathode end. All CE separations were conducted at 25 °C with a liquid thermostated capillary cartridge. A 50-μL Hamilton microsyringe with a fixed beveled-point needle was used for extraction. 2.2. Chemicals All electrolyte and standard solutions were prepared with doubly distilled helium degassed water. Imidazole (≥99.5%), 18crown-6 (≥99.5%), poly(diallyldimethylammonium chloride) (PDADMA, 20%, w/w, in water, average molecular mass ∼ 100,000–200,000) and poly(sodium-4-styrenesulfonate) (PSS, average molecular mass ∼70,000) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other reagents used for experiments detailed in this report were purchased from Merck (Darmstadt, Germany) and were of analytical-reagent grade.
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Standard solution of ammonium (0.01 mol/L) was prepared by dissolving ammonium chloride in water. Working ammonium solutions were prepared by diluting the standard solutions in water before use. Carrier electrolytes were prepared by neutralization of 5 mmol/L imidazole solution with acetic acid to desired pH. 2.3. Procedures Blood was drawn into a chilled vacuum tube that was immediately placed on ice, and ammonia was determined within 60 min. Milk was purchased from the market and the Baltic seawater sample was collected from the western coast of Lithuania. After the collection, samples were stored at 4 °C and ammonia was determined within 1 day. Prior to the analysis, milk sample was diluted 1:5 with doubly distilled water. Headspace SDME was carried out from 5 ml of sample solution placed in a 10 ml vial closed with the silicone rubber septum containing cap. Extraction was carried out as follows. A 4.0 ml sample solution and 1 ml of 0.5 mol/L potassium phosphate buffer (pH 12) were placed in a sample vial. After uptake of 5 μL of acceptor solution (aqueous solution containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4) the needle of the syringe was then inserted into the headspace of sample solution. The syringe plunger was depressed and a microdrop of acceptor phase was suspended from the needle tip. After an optimized period of time, the plunger was withdrawn and the microdrop was retracted back into the syringe. The needle was removed from the headspace and its content was introduced to a CE microvial for subsequent analysis. All electrolyte solutions were filtered through a 0.45 μm membrane filter degassed by ultrasonication. Each new fused silica capillary was flushed with 1 mol/L NaOH for 15 min and then with deionized water for 15 min. 3. Results and discussion 3.1. Choice of CE conditions Since the analyte studied exhibits any significant absorbance in the UV range, indirect detection mode at 214 nm using conventional imidazole containing carrier electrolyte was employed for CE separations [21]. The use of an internal standard is often necessary in order to compensate the relatively poor repeatability observed in SDME and CE techniques, and hence to achieve an acceptable method precision. For quantitative analysis, we have selected potassium cation as an internal standard. Potassium meets the main requirements for internal standard: it is compatible with the acceptor phase, chemically stable, nonvolatile, and has a sufficiently high mobility. Because the separation of NH4+ and K+ is difficult due to their identical electrophoretic mobilities at a neutral and acidic pH, 18-crown-6 was added to the carrier electrolyte. This crown ether reduces the mobility of potassium ion without any significant changes in the migration time for ammonium [22]. Thus, initial CE separations were performed in the carrier electrolyte containing 5 mmol/L of imidazole neutralized with CH3COOH to
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pH 4.3 and 3 mmol/L of 18-crown-6. Under the conventional CE conditions both cations were well resolved in about 3 min, but the peaks obtained were relatively broad and poorly shaped. In order to shorten analysis time and, consequently, to improve peak efficiency the electroosmotic flow (EOF) velocity was increased by a bilayer capillary coating with oppositely charged ionic polymers [23]. The capillary coating protocol used in this work involves flushing the capillary with 0.1% (w/v) aqueous PDADMA solution for 5 min followed by flushing the capillary with water for 1 min. Next, the capillary was flushed with 0.1% (w/v) aqueous PSS solution for 5 min. In addition, for best performance between all electrophoretic separations the capillary was rinsed with PSS solution for 1 min followed by flushing with carrier electrolyte for 1 min. Using this coating procedure, the second negatively charged PSS layer yielded very fast and stable cathodal electroosmotic flow. At pH 4.3 the cathodal EOF was about 5-times greater for coated capillary than for uncoated one. Fig. 1 shows the electropherograms of a test mixture of two cations separated under conventional CE conditions (Fig. 1a), and with a PDADMA/PSS-coated capillary (Fig. 1b). As can be observed, significantly better separation performance in respect to peak efficiency and separation time was obtained for coated capillary. 3.2. Extraction performance All the SDME experiments were performed with 5 ml of sample solution and 5 μL of aqueous acceptor phase containing
Fig. 1. Separation of ammonium and potassium in (a) the uncoated capillary and (b) the PDADMA/PSS coated capillary. Electrolyte, 5 mmol/L imidazole adjusted to pH 4.3 with CH3COOH, 3 mmol/L 18-crown-6; voltage, 25 kV; capillary, 50 cm (effective length) ×75 μm i.d.; detection, indirect UV at 214 nm; injection, hydrodynamic (3.43 × 103 Pa) for 10 s.
Fig. 2. Effect of sample pH on the extraction efficiency obtained for two ammonium concentrations. Extraction conditions: extraction time, 10 min; extraction temperature 25 °C. Acceptor phase, 1.0 mmol/L H3PO4 and 0.5 mmol/L KH2PO4. CE conditions as in Fig. 1b.
0.5 mmol/L KH2PO4 as an internal standard. It is well known that the agitation of the sample solution enhances the mass transfer in the sample solution and prompts the convection process in the headspace. This, eventually, leads to a shorter equilibrium time for the sample and the headspace and consequently a lowered extraction time, as well. For this reason all the extractions were performed at vigorous stirring rate of 800 rpm. Since ammonium cation is weakly acidic (pKa = 9.25), the pH of both the sample solution and the acceptor phase should be very important parameter in headspace SDME of ammonia. Basically, the sample solution should be alkaline enough in order to promote deprotonation of the weakly acidic NH4+ while the acceptor phase should be acidic in order to convert extracted
Fig. 3. Effect of temperature on the extraction efficiency.
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analyte into the nonvolatile ammonium ion. In addition, in order to avoid of the pH increase during extraction process due to coextraction of other volatile bases, the acceptor phase should have sufficiently high buffering capacity. Consequently, these effects must be taken into consideration in the design of optimum extraction conditions. The effect of sample pH was examined in the pH range between 8.0 and 13.0 by headspace extraction at room temperature for 10 min. Ammonium standards were diluted in 0.1 mol/L KH2PO4 electrolytes neutralized with KOH to desired pH. Three independent experiments for each pH value have been carried out. The results obtained for two different analyte concentrations (0.1 and 0.05 mmol/L) are demonstrated in Fig. 2. As expected, in the pH range 8.0–10, the extraction efficiency dramatically increases indicating that the deprotonation of the analyte takes place. The peak areas are maximum and do not depend on pH in the pH range 11–13. Therefore, a sample pH of 12 was selected for further experiments. The pH of the acceptor phase was varied in the pH range from 3 to 8 by neutralization of initial 1.0 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 solution with NaOH to desired pH. An increase of the acceptor phase pH from 3 to about 7 did not show any significant changes in the extraction efficiency. By further increase in the pH, a gradual decrease in extracted NH3 amount was observed due to reextraction of deprotonized ammonia from the drop. On the basis of the above experiments, aqueous solution containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 was selected as the acceptor phase for further studies. The second factor considered was the effect of temperature. The total extraction efficiency which is a compromise between the mass transfer of the analyte from the sample solution to the headspace and then from the headspace to the acceptor phase, is temperature-dependent. Higher temperatures lead to higher vapour pressure of the analyte and hence its concentration in headspace increases. On the other hand, the distribution
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Fig. 5. Electropherogram of blood sample. Extraction time, 15 min. For other conditions see Fig. 2.
constant of the ammonia between acceptor phase and headspace decreases with the temperature. The effect of temperature was studied in the range of 25–65 °C by exposing acceptor phase in the headspace for 10 min. Fig. 3 presents the effect of sample temperature on the extraction ability of NH3, obtained by plotting the relative peak area as a function of temperature. As can be seen, the extraction ability gradually increases, with increasing extraction temperature, up to 45 °C, due to the increasing distribution constant of analyte between the aqueous phase and headspace. However, a slight decrease in extracted NH3 amount was observed when the extraction temperature was set higher than about 50–55 °C. Furthermore, when extraction temperature was set at 70 °C or higher, the microdrop became unstable and tended to detach from the needle tip. Although the maximum peak areas were obtained at 45–55 °C, room temperature was adopted to ensure a stable, more reproducible and simple operation without decreasing the sensitivity of the method significantly. SDME is an equilibrium-based technique and there is a direct relationship between the amount extracted and the microdrop exposure time in the headspace of the sample. In order to obtain better repeatability and higher sensitivity of the analysis an equilibrium between the sample, headspace and the acceptor phase should be reached. Extraction time effect was studied in the range of 5–30 min at 25 °C. A plot of relative peak area versus extraction time (Fig. 4) shows that ammonia amount Table 1 Results of the ammonium in real samples (n = 3)
Fig. 4. Effect of extraction time on the extraction efficiency.
Sample
Found, μmol/L mean ± SD a
Added, μmol/L
Found total, μmol/L mean ± SD
Recovery (%)
Human blood
34.1 ± 2.7
Seawater
18.3 ± 1.0
Milk
262 ± 16
10.0 25.0 10.0 25.0 50.0 125
44.8 ± 3.2 59.7 ± 2.9 28.1 ± 1.6 43.0 ± 2.6 310 ± 14 384 ± 19
107.1 102.4 98.0 98.8 96.0 97.6
a
Standard deviation.
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extracted increases with sampling time in the range of 5– 15 min, reaches equilibrium after about 15 min, and then stays almost constant until 30 min. Therefore, an extraction time of 15 min was chosen for further studies.
Acknowledgement
3.3. Analytical performance
References
To evaluate the practical applicability of the proposed SDME technique, several analytical performance characteristics such as enrichment factor, linearity, limit of detection (LOD) and repeatability were investigated under optimized conditions. The enrichment factor, defined as the ratio of relative peak areas after extraction and that before extraction, was used to evaluate the extraction efficiency. With headspace SDME at room temperature for 15 min ammonia was enriched by a factor of approximately 14. The calibration curve was linear for concentrations of NH4+ in the range from 5 to 100 μmol/L (R2 = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/ L of NH4+. The relative standard deviations (RSDs) obtained after six consecutive extractions of ammonia standard at two concentration levels (10 and 50 μmol/L) were calculated to be 7.5 and 5.3%, respectively. The repeatability was acceptable and comparable with other microextraction techniques reported in the literature [12]. These data support the suitability of the proposed method for its application to common environmental and biological samples. To finish the evaluation of headspace SDME method, ammonia was determined in human blood, seawater and milk samples. Typical electropherogram of the blood sample is shown in Fig. 5 and the results obtained are summarized in Table 1. The method accuracy was validated by spiking natural samples with known amounts of ammonia and evaluating the recovery (Table 1). For all samples the t-test performed at the 5% significance level demonstrated that there was no statistically significant difference between expected and found values. Obtained results showed that the sample matrices did not interfere with the determination of ammonia.
[1] J.R. Huizenga, A. Tangerman, Gips, determination of ammonia in biological fluids, Ann. Clin. Biochem. 31 (1994) 529–543. [2] R.J. Barsotti, Measurement of ammonia in blood, J. Pediatr. 138 (2001) S11–S20. [3] B. Timmer, W. Olthuis, A. van den Berg, Ammonia sensors and their applications — a review, Sens. Actuators, B, Chem. 107 (2005) 666–677. [4] S.B. Shah, P.W. Westerman, J. Arongo, Measuring ammonia concentrations and emissions from agricultural land and liquid surfaces: a review, J. Air Waste Manage. Assoc. 56 (2006) 945–960. [5] P.V.D. Burg, H.W. Mook, A simple and rapid method for the determination of ammonia in blood, Clin. Chim. Acta 8 (1963) 162–164. [6] E. Conway, A. Byrne, An absorption apparatus for the micro-determination of certain volatile substances. The micro-determination of ammonia, Biochem. J. 27 (1933) 419–429. [7] S.G. Dienst, An ion exchange method for plasma ammonia concentration, J. Lab. Clin. Med. 58 (1961) 149–155. [8] Y. Saito, K. Jinno, Miniaturized sample preparation combined with liquid phase separations, J. Chromatogr., A 1000 (2003) 53–67. [9] H. Liu, P.K. Dasgupta, Analytical chemistry in a drop. Solvent extraction in a microdrop, Anal. Chem. 68 (1996) 1817–1821. [10] M.A. Jeannot, F.F. Cantwell, Solvent microextraction into a single drop, Anal. Chem. 68 (1996) 2236–2240. [11] Y. He, H.K. Lee, Liquid-phase microextraction in a single drop of organic solvent by using a conventional microsyringe, Anal. Chem. 69 (1997) 4634–4640. [12] E. Psillakis, N. Kalogerakis, Developments in single-drop microextraction, Trends Anal. Chem. 21 (2002) 53–63. [13] A.L. Theis, A.J. Waldack, S.M. Hansen, M.A. Jeannot, Headspace solvent microextraction, Anal. Chem. 73 (2001) 5651–5654. [14] A. Tankeviciute, R. Kazlauskas, V. Vickackaite, Headspace extraction of alcohols into a single drop, Analyst 126 (2001) 1674–1677. [15] A. Przyjazny, J.M. Kokosa, Analytical characteristics of the determination of benzene, toluene, ethylbenzene and xylenes in water by headspace solvent microextraction, J. Chromatogr., A 977 (2002) 143–153. [16] M.R. Milani, J.A.C. Neto, A.A. Cardoso, Colorimetric determination of sulfur dioxide in air using a droplet collector of malachite green solution, Microchem. J. 62 (1999) 273–281. [17] A.A. Cardoso, H. Liu, P.K. Dasgupta, Fluorometric fiber optic drop sensor for atmospheric hydrogen sulfide, Talanta 44 (1997) 1099–1106. [18] A. Pretto, M.R. Milani, A.A. Cardoso, Colorimetric determination of formaldehyde in air using a hanging drop of chromotrophic acid, J. Environ. Monit. 2 (2001) 566–570. [19] V. Colombini, C. Bancon-Montigny, L. Yang, P. Maxwell, R.E. Sturgeon, Z. Mester, Headspace single-drop microextration for the detection of organotin compounds, Talanta 63 (2004) 555–560. [20] N. Li, C. Deng, X. Yin, N. Yao, X. Shen, X. Zhang, Gas chromatography– mass spectrometric analysis of hexanal and heptanal in human blood by headspace single-drop microextraction with droplet derivatization, Anal. Biochem. 342 (2005) 318–326. [21] W. Beck, H. Engelhardt, Capillary electrophoresis of organic and inorganic cations with indirect UV detection, Chromatographia 33 (1992) 313–316. [22] A. Padarauskas, V. Paliulionyte, B. Pranaityte, Single-run capillary electrophoretic determination of inorganic nitrogen species in rainwater, Anal. Chem. 73 (2001) 267–271. [23] B. Pranaityte, A. Padarauskas, Micellar electrokinetic chromatography at low pH with polyelectrolyte-coated capillaries, J. Chromatogr., A 1042 (2004) 197–202.
4. Conclusion This study shows that water-based headspace SDME and subsequent capillary electrophoretic analysis is a useful technique which complements existing methodologies in the analysis of ammonia in various environmental and biological samples. The main advantage of this method is that sample clean-up and preconcentration procedures can be completed in a single step. Macromolecules and most small organic and inorganic compounds are nonvolatile under extraction conditions employed and therefore remained in the sample solution. The main problem in the determination of ammonia in biological samples is the formation additional ammonia during sample pretreatment (deproteinization, alkaline distillation, derivatization, etc.) procedures. Using rapid and simple headspace SDME technique this problem was completely avoided.
This work has been supported by the Lithuanian State Science and Studies Foundation.
Capillary electrophoretic determination of ammonia using headspace single-drop microextraction Birutė Pranaitytė a , Svetlana Jermak a , Evaldas Naujalis b , Audrius Padarauskas a,⁎ a
Department of Analytical and Environmental Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania Laboratory for Metrology in Chemistry, Semiconductor Physics Institute, A. Gostauto 11, LT-01108 Vilnius, Lithuania
b
Received 28 August 2006; received in revised form 10 October 2006; accepted 10 October 2006 Available online 17 November 2006
Abstract A new method involving headspace single-drop microextraction (SDME) and capillary electrophoresis (CE) is developed for the preconcentration and determination of ammonia (as dissolved NH3 and ammonium ion). An aqueous microdrop (5 μL) containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 (as internal standard) was used as the acceptor phase. Common experimental parameters (sample and acceptor phase pH, extraction temperature, extraction time) affecting the extraction efficiency were investigated. Proposed SDME-CE method provided about 14-fold enrichment in about 20 min. The calibration curve was linear for concentrations of NH+4 in the range from 5 to 100 μmol/L (R2 = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/L of NH+4 . Such detection sensitivity is high enough for ammonia determination in common environmental and biological samples. Finally, headspace SDME was applied to determine ammonia in human blood, seawater and milk samples with spiked recoveries in the range of 96–107%. © 2006 Elsevier B.V. All rights reserved. Keywords: Single-drop microextraction; Capillary electrophoresis; Ammonia
1. Introduction Ammonia is a widely occurring natural compound, often produced as a consequence of microbial protein catabolism, and thus serves as a quality indicator of fruit juice, milk, cheese, meat, seafood, and bakery products. In the wine industry, ammonia determination is important in the calculation of yeast available nitrogen. It is also monitored in swimming pool water, waste water, and fermentation cultures, where its detection may indicate the presence of faeces, urine and microbes. An elevated ammonia blood level is considered a strong indicator of an abnormality in nitrogen homeostasis, the most common related to liver dysfunction. Ammonia is also an important micronutrient and intermediate of the nitrogen cycle in aquatic ecosystem. High concentrations of ammonia in water can lead to nutrient enrichment of water systems, and the subsequent increase in biological activity can lead to algal blooms the potential for turbidity, taste, odor and toxicity problems. There⁎ Corresponding author. Fax: +370 5 2330987. E-mail address: [email protected] (A. Padarauskas). 0026-265X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2006.10.001
fore, the development of a rapid and simple analytical technique for the monitoring of ammonia (as dissolved NH3 and ammonium ion) in various samples is of significant interest. There are many methods used for the determination of ammonia or ammonium ion [1–4]. However, the determination of ammonia in the samples with complicated matrices requires the separation of ammonia from sample matrix which is usually achieved through alkaline distillation [5], aeration/microdiffusion [6] or solid-phase extraction [7]. Each method has its own merits, but they are laborious, time-consuming and often lack sensitivity. In the past decade, one noticeable trend in the development of sample preparation techniques has been miniaturization [8]. Miniaturized modification of the traditional liquid–liquid extraction method termed single-drop microextraction (SDME) is one of the recent trends in this area [9–11]. In practice, two main approaches can be used to perform single-drop microextractions. In direct single-drop microextraction, a drop of a water-immiscible solvent is suspended directly from the tip of a microsyringe needle immersed in the aqueous sample. This extraction approach, in combination with GC or
B. Pranaitytė et al. / Microchemical Journal 86 (2007) 48–52
HPLC, has been shown to be quite effective for extraction and analysis of various organic compounds [12]. However, applications of direct SDME are normally restricted to medium polarity and non-polar analytes and those whose polarities can be reduced before extraction. Furthermore, the organic solvents used for direct SDME which are immiscible with water, are not compatible with conventional capillary electrophoresis (CE). An alternative way of minimizing some of these problems is to use headspace SDME. In this approach a microdrop of appropriate solvent is placed in the headspace of the sample solution [13–15] or in a flowing air sample stream [9,16–18] to extract volatile analytes. Headspace SDME has similar capabilities in terms of precision and speed of analysis compared to direct SDME but has an advantage to choice of a wider variety of solvents. Unlike direct SDME, water can be also used as the solvent to extract volatile and water soluble analytes. This significantly enhances the range extractable analytes as well as the range of analytical methods that can be coupled with SDME. In addition, headspace SDME was found to provide excellent clean-up from the samples with complicated matrices [19,20]. The aim of this work was to provide a new approach for the rapid and simple monitoring of ammonia involving water-based headspace single-drop microextraction with subsequent capillary electrophoretic determination. The feasibility of the method was demonstrated by the analysis of human blood, milk and seawater samples. 2. Experimental 2.1. Instrumentation Separations were performed on a P/ACE 2100 apparatus (Beckman Instruments Inc., Fullerton, CA, USA) equipped with a UV detector with wavelength filters (200, 214, 230 and 254 nm). Fused silica capillaries (Polymicro Technology, Phoenix, AZ, USA) of 75 μm i.d. and 57 cm total length (50 cm to the detector) were used. Samples were injected in the hydrodynamic mode by overpressure (3.43 × 103 Pa). System Gold software (Beckman Instruments Inc.) was used for data acquisition. Indirect UV detection (214 nm) was employed at the cathode end. All CE separations were conducted at 25 °C with a liquid thermostated capillary cartridge. A 50-μL Hamilton microsyringe with a fixed beveled-point needle was used for extraction. 2.2. Chemicals All electrolyte and standard solutions were prepared with doubly distilled helium degassed water. Imidazole (≥99.5%), 18crown-6 (≥99.5%), poly(diallyldimethylammonium chloride) (PDADMA, 20%, w/w, in water, average molecular mass ∼ 100,000–200,000) and poly(sodium-4-styrenesulfonate) (PSS, average molecular mass ∼70,000) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other reagents used for experiments detailed in this report were purchased from Merck (Darmstadt, Germany) and were of analytical-reagent grade.
49
Standard solution of ammonium (0.01 mol/L) was prepared by dissolving ammonium chloride in water. Working ammonium solutions were prepared by diluting the standard solutions in water before use. Carrier electrolytes were prepared by neutralization of 5 mmol/L imidazole solution with acetic acid to desired pH. 2.3. Procedures Blood was drawn into a chilled vacuum tube that was immediately placed on ice, and ammonia was determined within 60 min. Milk was purchased from the market and the Baltic seawater sample was collected from the western coast of Lithuania. After the collection, samples were stored at 4 °C and ammonia was determined within 1 day. Prior to the analysis, milk sample was diluted 1:5 with doubly distilled water. Headspace SDME was carried out from 5 ml of sample solution placed in a 10 ml vial closed with the silicone rubber septum containing cap. Extraction was carried out as follows. A 4.0 ml sample solution and 1 ml of 0.5 mol/L potassium phosphate buffer (pH 12) were placed in a sample vial. After uptake of 5 μL of acceptor solution (aqueous solution containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4) the needle of the syringe was then inserted into the headspace of sample solution. The syringe plunger was depressed and a microdrop of acceptor phase was suspended from the needle tip. After an optimized period of time, the plunger was withdrawn and the microdrop was retracted back into the syringe. The needle was removed from the headspace and its content was introduced to a CE microvial for subsequent analysis. All electrolyte solutions were filtered through a 0.45 μm membrane filter degassed by ultrasonication. Each new fused silica capillary was flushed with 1 mol/L NaOH for 15 min and then with deionized water for 15 min. 3. Results and discussion 3.1. Choice of CE conditions Since the analyte studied exhibits any significant absorbance in the UV range, indirect detection mode at 214 nm using conventional imidazole containing carrier electrolyte was employed for CE separations [21]. The use of an internal standard is often necessary in order to compensate the relatively poor repeatability observed in SDME and CE techniques, and hence to achieve an acceptable method precision. For quantitative analysis, we have selected potassium cation as an internal standard. Potassium meets the main requirements for internal standard: it is compatible with the acceptor phase, chemically stable, nonvolatile, and has a sufficiently high mobility. Because the separation of NH4+ and K+ is difficult due to their identical electrophoretic mobilities at a neutral and acidic pH, 18-crown-6 was added to the carrier electrolyte. This crown ether reduces the mobility of potassium ion without any significant changes in the migration time for ammonium [22]. Thus, initial CE separations were performed in the carrier electrolyte containing 5 mmol/L of imidazole neutralized with CH3COOH to
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pH 4.3 and 3 mmol/L of 18-crown-6. Under the conventional CE conditions both cations were well resolved in about 3 min, but the peaks obtained were relatively broad and poorly shaped. In order to shorten analysis time and, consequently, to improve peak efficiency the electroosmotic flow (EOF) velocity was increased by a bilayer capillary coating with oppositely charged ionic polymers [23]. The capillary coating protocol used in this work involves flushing the capillary with 0.1% (w/v) aqueous PDADMA solution for 5 min followed by flushing the capillary with water for 1 min. Next, the capillary was flushed with 0.1% (w/v) aqueous PSS solution for 5 min. In addition, for best performance between all electrophoretic separations the capillary was rinsed with PSS solution for 1 min followed by flushing with carrier electrolyte for 1 min. Using this coating procedure, the second negatively charged PSS layer yielded very fast and stable cathodal electroosmotic flow. At pH 4.3 the cathodal EOF was about 5-times greater for coated capillary than for uncoated one. Fig. 1 shows the electropherograms of a test mixture of two cations separated under conventional CE conditions (Fig. 1a), and with a PDADMA/PSS-coated capillary (Fig. 1b). As can be observed, significantly better separation performance in respect to peak efficiency and separation time was obtained for coated capillary. 3.2. Extraction performance All the SDME experiments were performed with 5 ml of sample solution and 5 μL of aqueous acceptor phase containing
Fig. 1. Separation of ammonium and potassium in (a) the uncoated capillary and (b) the PDADMA/PSS coated capillary. Electrolyte, 5 mmol/L imidazole adjusted to pH 4.3 with CH3COOH, 3 mmol/L 18-crown-6; voltage, 25 kV; capillary, 50 cm (effective length) ×75 μm i.d.; detection, indirect UV at 214 nm; injection, hydrodynamic (3.43 × 103 Pa) for 10 s.
Fig. 2. Effect of sample pH on the extraction efficiency obtained for two ammonium concentrations. Extraction conditions: extraction time, 10 min; extraction temperature 25 °C. Acceptor phase, 1.0 mmol/L H3PO4 and 0.5 mmol/L KH2PO4. CE conditions as in Fig. 1b.
0.5 mmol/L KH2PO4 as an internal standard. It is well known that the agitation of the sample solution enhances the mass transfer in the sample solution and prompts the convection process in the headspace. This, eventually, leads to a shorter equilibrium time for the sample and the headspace and consequently a lowered extraction time, as well. For this reason all the extractions were performed at vigorous stirring rate of 800 rpm. Since ammonium cation is weakly acidic (pKa = 9.25), the pH of both the sample solution and the acceptor phase should be very important parameter in headspace SDME of ammonia. Basically, the sample solution should be alkaline enough in order to promote deprotonation of the weakly acidic NH4+ while the acceptor phase should be acidic in order to convert extracted
Fig. 3. Effect of temperature on the extraction efficiency.
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analyte into the nonvolatile ammonium ion. In addition, in order to avoid of the pH increase during extraction process due to coextraction of other volatile bases, the acceptor phase should have sufficiently high buffering capacity. Consequently, these effects must be taken into consideration in the design of optimum extraction conditions. The effect of sample pH was examined in the pH range between 8.0 and 13.0 by headspace extraction at room temperature for 10 min. Ammonium standards were diluted in 0.1 mol/L KH2PO4 electrolytes neutralized with KOH to desired pH. Three independent experiments for each pH value have been carried out. The results obtained for two different analyte concentrations (0.1 and 0.05 mmol/L) are demonstrated in Fig. 2. As expected, in the pH range 8.0–10, the extraction efficiency dramatically increases indicating that the deprotonation of the analyte takes place. The peak areas are maximum and do not depend on pH in the pH range 11–13. Therefore, a sample pH of 12 was selected for further experiments. The pH of the acceptor phase was varied in the pH range from 3 to 8 by neutralization of initial 1.0 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 solution with NaOH to desired pH. An increase of the acceptor phase pH from 3 to about 7 did not show any significant changes in the extraction efficiency. By further increase in the pH, a gradual decrease in extracted NH3 amount was observed due to reextraction of deprotonized ammonia from the drop. On the basis of the above experiments, aqueous solution containing 1 mmol/L H3PO4 and 0.5 mmol/L KH2PO4 was selected as the acceptor phase for further studies. The second factor considered was the effect of temperature. The total extraction efficiency which is a compromise between the mass transfer of the analyte from the sample solution to the headspace and then from the headspace to the acceptor phase, is temperature-dependent. Higher temperatures lead to higher vapour pressure of the analyte and hence its concentration in headspace increases. On the other hand, the distribution
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Fig. 5. Electropherogram of blood sample. Extraction time, 15 min. For other conditions see Fig. 2.
constant of the ammonia between acceptor phase and headspace decreases with the temperature. The effect of temperature was studied in the range of 25–65 °C by exposing acceptor phase in the headspace for 10 min. Fig. 3 presents the effect of sample temperature on the extraction ability of NH3, obtained by plotting the relative peak area as a function of temperature. As can be seen, the extraction ability gradually increases, with increasing extraction temperature, up to 45 °C, due to the increasing distribution constant of analyte between the aqueous phase and headspace. However, a slight decrease in extracted NH3 amount was observed when the extraction temperature was set higher than about 50–55 °C. Furthermore, when extraction temperature was set at 70 °C or higher, the microdrop became unstable and tended to detach from the needle tip. Although the maximum peak areas were obtained at 45–55 °C, room temperature was adopted to ensure a stable, more reproducible and simple operation without decreasing the sensitivity of the method significantly. SDME is an equilibrium-based technique and there is a direct relationship between the amount extracted and the microdrop exposure time in the headspace of the sample. In order to obtain better repeatability and higher sensitivity of the analysis an equilibrium between the sample, headspace and the acceptor phase should be reached. Extraction time effect was studied in the range of 5–30 min at 25 °C. A plot of relative peak area versus extraction time (Fig. 4) shows that ammonia amount Table 1 Results of the ammonium in real samples (n = 3)
Fig. 4. Effect of extraction time on the extraction efficiency.
Sample
Found, μmol/L mean ± SD a
Added, μmol/L
Found total, μmol/L mean ± SD
Recovery (%)
Human blood
34.1 ± 2.7
Seawater
18.3 ± 1.0
Milk
262 ± 16
10.0 25.0 10.0 25.0 50.0 125
44.8 ± 3.2 59.7 ± 2.9 28.1 ± 1.6 43.0 ± 2.6 310 ± 14 384 ± 19
107.1 102.4 98.0 98.8 96.0 97.6
a
Standard deviation.
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extracted increases with sampling time in the range of 5– 15 min, reaches equilibrium after about 15 min, and then stays almost constant until 30 min. Therefore, an extraction time of 15 min was chosen for further studies.
Acknowledgement
3.3. Analytical performance
References
To evaluate the practical applicability of the proposed SDME technique, several analytical performance characteristics such as enrichment factor, linearity, limit of detection (LOD) and repeatability were investigated under optimized conditions. The enrichment factor, defined as the ratio of relative peak areas after extraction and that before extraction, was used to evaluate the extraction efficiency. With headspace SDME at room temperature for 15 min ammonia was enriched by a factor of approximately 14. The calibration curve was linear for concentrations of NH4+ in the range from 5 to 100 μmol/L (R2 = 0.996). The LOD (S / N = 3) was estimated to be 1.5 μmol/ L of NH4+. The relative standard deviations (RSDs) obtained after six consecutive extractions of ammonia standard at two concentration levels (10 and 50 μmol/L) were calculated to be 7.5 and 5.3%, respectively. The repeatability was acceptable and comparable with other microextraction techniques reported in the literature [12]. These data support the suitability of the proposed method for its application to common environmental and biological samples. To finish the evaluation of headspace SDME method, ammonia was determined in human blood, seawater and milk samples. Typical electropherogram of the blood sample is shown in Fig. 5 and the results obtained are summarized in Table 1. The method accuracy was validated by spiking natural samples with known amounts of ammonia and evaluating the recovery (Table 1). For all samples the t-test performed at the 5% significance level demonstrated that there was no statistically significant difference between expected and found values. Obtained results showed that the sample matrices did not interfere with the determination of ammonia.
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4. Conclusion This study shows that water-based headspace SDME and subsequent capillary electrophoretic analysis is a useful technique which complements existing methodologies in the analysis of ammonia in various environmental and biological samples. The main advantage of this method is that sample clean-up and preconcentration procedures can be completed in a single step. Macromolecules and most small organic and inorganic compounds are nonvolatile under extraction conditions employed and therefore remained in the sample solution. The main problem in the determination of ammonia in biological samples is the formation additional ammonia during sample pretreatment (deproteinization, alkaline distillation, derivatization, etc.) procedures. Using rapid and simple headspace SDME technique this problem was completely avoided.
This work has been supported by the Lithuanian State Science and Studies Foundation.