Equilibrium Sampling through Membranes of ... - ACS Publications

Anal. Chem. 2005, 77, 4800-4809

Equilibrium Sampling through Membranes of Freely Dissolved Chlorophenols in Water Samples with Hollow Fiber Supported Liquid Membrane Jing-fu Liu,† Jan Åke Jo 1 nsson,*,† and Philipp Mayer‡

Department of Analytical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden, and National Environmental Research Institute, P.O. Box 358, 4000 Roskilde, Denmark

The freely dissolved concentration (Cfree) of pollutants is generally believed to be bioavailable and thus responsible for toxic effects. The Cfree of organic weak acids and bases consists of a dissociated and a nondissociated fraction. By using chlorophenols as model compounds, a negligibledepletion extraction technique, equilibrium sampling through membranes (ESTM), was developed for the measurement of the nondissociated part of the Cfree. Polypropylene hollow fiber membranes (280-µm i.d., 50µm wall thickness, 0.1-µm pore size, 15-cm length) were impregnated with undecane in the pores in the fiber wall as liquid membrane and filled with buffer solution in the lumen as acceptor. Then, the hollow fiber membranes were placed into the sample (donor) for an equilibrium extraction after sealing the two ends. The chlorophenol concentrations in the acceptor were then determined by direct injection into a HPLC system. Finally, the Cfree of the nondissociated and the dissociated species of a chlorophenol were calculated based on its measured concentration in the acceptor, its pKa value, and the measured pH in sample and acceptor. Theoretically calculated distribution coefficients (D ) 8-970) agree well with the experimental enrichment factors (Ee(max) ) 6-1124), and the equilibration time was observed to increase with increasing distribution coefficients (hours to days). The freely dissolved concentration of five chlorophenols, with a wide range of pKa (4.9-9.2) and log Kow (2.35-5.24), were successfully determined in model solutions of humic acids and at low-ppb levels in river and leachate water. Freely dissolved concentration (Cfree) of a chemical is believed to be the driving force for the transport, distribution, and bioaccumulation1 and thus a key parameter controlling the bioavailability and the toxic effect of a pollutant in the environment.2 Therefore, the determination of free concentration is of great interest and many methods are developed for this purpose. Heringa and Hermens3 presented a comprehensive review on the * To whom correspondence should be addressed. E-mail: jan_ake.jonsson@ analykem.lu.se. Fax: +46 46 222 45 44. Tel: +46 46 222 81 69. † Lund University. ‡ National Environmental Research Institute. (1) Mackay, D.; Paterson, S. Environ. Sci. Technol. 1991, 25, 427. (2) Heringa, M. B.; Hermens, J. L. M. Trends Anal. Chem. 2003, 22, 575-585.

4800 Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

presently available procedures for the determination of Cfree. For environmental purposes, the most commonly used methods for sensing Cfree of pollutants include diffusion gradients in thin films (DGT)4 for metals, as well as semipermeable membrane devices (SPMDs),5 and solid-phase microextraction (SPME)6-8 for organic compounds, especially hydrophobic organic compounds. While DGT and SPMDs are generally operated in the kinetic uptake regime as they need a long time to reach equilibrium, SPME was successfully used as an equilibrium sampling device due to its relatively short equilibration time.9 Although SPME has been applied for sampling various compounds, it suffers from low extraction efficiency for some polar analytes and it is not quite compatible with high-performance liquid chromatography (HPLC). Therefore, development of equilibrium sampling devices that facilitate sampling of Cfree of relatively hydrophilic compounds in a complex matrix is of great importance. Solvent microextraction10 based on suspension of a 1-µL drop of n-octane on the tip of a microsyringe needle in 500 mL of aqueous solution, which was proposed to sample the unbound progesterone in the presence of bovine serum albumin (water-soluble macromolecule), has the potential to extract polar compounds. However, this technique suffers from the solvent drop being vulnerable to physical forces. Supported liquid membrane (SLM) extraction, with flat membrane or hollow fiber membrane as supporting material for an organic solvent to form an aqueous-organic solvent-aqueous three-phase system, has been applied to sampling of various compounds, including weak acids and bases, as well as metal ions.11,12 At present, almost all the SLM-based sampling applica(3) Suffet, I. H.; Javfert, C. T.; Kukkonen, J.; Servos, M. R.; Spacie, A.; Williams, L. L.; Noblet, J. A. Synopsis of discussion session: Influences of particulate and dissolved material on the bioavailability of organic compounds; CRC Press (Lewis Publishers): Boca Raton, FL, 1994; pp 93-107. (4) Davison, W.; Zhang, H. Nature 1994, 367, 546-548. (5) Petty, J. D.; Poulton, B. C.; Charbonneau, C. S.; Huckins, J. N.; Jones, S. B.; Cameron, J. T.; Prest, H. F. Environ. Sci. Technol. 1998, 32, 837-842. (6) Vaes, W. H. J.; Urrestarazu Ramos, E.; Verhaar, H. J. M.; Seinen, W.; Hermens, J. L. M. Anal. Chem. 1996, 68, 4463-4467. (7) Poerschmann, J.; Kopinke, F.-D.; Pawliszyn, J. Environ. Sci. Technol. 1997, 31, 3629-3636. (8) Poerschmann, J.; Zhang, Z.; Kopinke, F.-D.; Pawliszyn, J. Anal. Chem. 1997, 69, 597-600. (9) Mayer, P.; Tolls, J.; Hermens, J. L. M.; Mackay, D. Environ. Sci. Technol. 2003, 37, 185A-191A. (10) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1997, 69, 2935-2940. (11) Jo ¨nsson, J. Å.; Mathiasson, L. J. Chromatogr., A 2000, 902, 205-225. (12) Rasmussen, K. E.; Pedersen-Bjergaard, S. Trends Anal. Chem. 2004, 23, 1-10. 10.1021/ac0503512 CCC: $30.25

© 2005 American Chemical Society Published on Web 06/15/2005

tions were performed in the kinetic uptake regime; i.e., equilibrium is far from being attained; and most of the applications aim at complete extractions and the measurement of total concentrations, i.e., to extract as much of the analyte as possible by manipulating the chemical conditions of the sample such as changing pH, adding various extractants or ion pair formers, etc. For example, in a recent study on SLM extraction of phenols by Lee et al.,13 samples were acidified to obtain high enrichment factor, i.e., to extract as much of the analyte as possible. These manipulations, however, influence the speciation (charged/uncharged) and the availability (bound/freely dissolved) of target analytes and therefore prohibit the measurement of specific chemical species or the direct measurement of Cfree. However, SLM has the potential to be used as an equilibrium sampling technique for measurement of Cfree through the following adjustment: (1) Select appropriate membrane and acceptor in order to attain equilibrium within a reasonable time, i.e., employ the “incomplete trapping” mode of operation of SLM;14 (2) Limit the “manipulation” of SLM to the membrane and the acceptor solution, while preserving the chemical composition and thus speciation and sorption equilibria in the sample. (3) Adopt a sample volume that is sufficiently large to ensure “negligibledepletion” sampling; i.e., the extracted amount was kept below 5% of the freely dissolved amount.6,9 The resulting sampling technique is termed equilibrium sampling through membranes (ESTM). Recently, this technique, based on flat membranes, has been applied to sampling of free Cu concentrations in environmental water15 and of free drug concentrations in pharmaceutical applications.16 In this present study, a hollow fiber membrane SLM extraction technique was adapted to ESTM sampling of five chlorophenols (CPs) within a wide range of pKa (4.9-9.2) and log Kow (2.355.24). The hollow fiber membranes were impregnated with undecane, and the lumen was filled with buffer solution as acceptor. Then the two ends of the fiber were sealed, and the whole fiber was put into the sample for extracting the uncharged species of freely dissolved CPs. After reaching equilibrium, the chlorophenol concentration in the acceptor was measured by HPLC. The Cfree of undissociated species and of dissociated ions were then separately obtained based on the pKa values, sample pH, and appropriate calibration.

Additionally, there is a dissociation equilibrium for the HAfree that is pH dependent:

HAfree / H+ + Afree-

Also, for the dissociated form, an equilibrium between the freely dissolved form and a bound form, Abound- should be considered. The total freely dissolved concentration (Cfree) is then the sum of both free forms:

Cfree ) [HA]free + [A-]free

Determination of Cfree Using ESTM. For a nondissociated form of a weak acid (HA) that is present in an environmental matrix, equilibrium between the freely dissolved form, HAfree, and the bound form, HAbound is established:

HAfree / HAbound

(1)

(13) Jiang, X.; Oh, S. Y.; Lee, H. K., Anal. Chem. 2005, 77, 1689-1695. (14) Chimuka, L.; Megersa, N.; Norberg, J.; Mathiasson, L.; Jo¨nsson, J. Å. Anal. Chem. 1998, 70, 3906-3911. (15) Romero, R.; Jo ¨nsson, J. Å. Anal. Bioanal. Chem. 2005, 381, 1452-1459. (16) Trtic´-Petrovic´, T.; Jo ¨nsson, J. Å. J. Chromatogr., B 2005, 814, 375-384.

(3)

From here, the subscript “free” is in most cases dropped for simplicity; if not otherwise stated, all concentrations in the sample refer to freely dissolved concentrations. The SLM extraction system consists of three immiscible phases (aq/org/aq): (1) donor, which is the aqueous sample to be extracted, (2) membrane, which is an organic liquid held in the pores of a hydrophobic polymer, and (3) acceptor, which is an aqueous buffer.11 Only the nondissociated HA can be transported from the donor, through the membrane, and be trapped in the acceptor. This extraction process is driven by the concentration difference of HA over the membrane until reaching equilibrium, when the following relation holds:

[HA] ) [HA]AKA/KD ) CARAKA/KD

(4)

Here, CA is the total concentration of analyte in the acceptor, KA and KD are the partition coefficients (to the organic phase) for the analyte in the acceptor and donor phases, respectively, and RA is the nondissociated fraction in the acceptor given by

RA )

1 1 + 10(pHA-pKa)

(5)

where pKa is the acid dissociation constant and pHA is the pH in the acceptor. Assuming that KA ) KD, [HA] in the sample can be determined based on the measured equilibrium concentration in the acceptor (CA), the pKa of the analyte, and the pH of the acceptor. Further, the total free concentration (Cfree) as well as [A-] can be derived based on [HA] and the pH of the sample (cf. eq 3):

Cfree ) CD ) [HA]/RD

THEORY

(2)

(6)

where RD is the nondissociated fraction in the sample and is calculated analogously to eq 5. Finally, the total analyte concentration can be determined with an exhaustive extraction technique. The total concentration will include free and bound forms and will then lead to a complete characterization of the speciation of organic, dissociable compounds. Evaluation of the ESTM Technique. In equilibrium sampling, the enrichment factor is governed by the distribution coefficient (D) between the donor and the acceptor, and a characteristic of negligible depletion sampling is that the concenAnalytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Table 1. Dissociation Constants (pKa), Octanol/Water Partition Coefficients (log Kow), and Calculated Distribution Coefficients (D) at Equilibrium Acceptor pH 9.60 and Various Sample (Donor) pH of the Five Studied Chlorophenols D at various donor pH compound 4-chlorophenol 2,5-dichlorophenol 2,4,5-trichlorophenol 2,3,5,6-tetrachlorophenol pentachlorophenol c

abbrev

pKa

log Kow

5.00

6.00

6.50

7.00

7.60

8.00

8.60

CP DCP TrCP TeCP PCP

9.2a

2.35d

7.5b 6.9c 5.5a 4.9a

3.20d 3.71c 4.06b 5.24c

3.5 127 495 9566 22186

3.5 123 445 3024 3688

3.5 115 358 1144 1228

3.5 96 222 386 395

3.4 56 83 99 100

3.3 30 37 40 40

2.9 9.3 9.8 10 10

a Value from ref 23. b Value calculated using a computer program, ACD/Labs for Chemistry, Advanced Chemistry Development Inc., Canada. Value from ref 24. d Value from ref 25.

tration in the sample is preserved throughout the sampling process. Under these conditions, distribution coefficients can be applied as calibration factors, and it is therefore crucial to evaluate how accurately D values can be predicted.

D ) CA/CD ) (RDKD)/(RAKA)

(7)

Knowing pHD and pHA as well as pKa and assuming that KD equals KA, which is the ideal situation, it is possible to calculate D, which is used to compare with experimental data. In real sampling, KD might be unequal to KA due to, for example, effects of different ionic strengths in the sample and in the acceptor buffer. Thus, there will be a difference between the experimental enrichment factor (Ee ) CA/CD) at equilibrium, Ee(max), and the calculated D value. Much of the experiments presented here aim at thorough studies of these relations at environmentally relevant conditions, with the intention to develop a calibration strategy that allows the calculation of the freely dissolved concentration Cfree from the determined CA and eqs 7 and 6. Calibration Method. Experiments of this study demonstrated that the Ee(max)/D ratio is independent of the buffer capacity, salinity, and pH of a sample solution. Therefore, the Ee(max)/D ratio was adopted for calibration in the following way: (1) the D value was calculated (using eq 7) based on the measured equilibrium pH of sample (pHD) and acceptor (pHA) and the most reliable value for pKa; (2) the Ee(max) value was calculated based on the constant Ee(max)/D ratio; (3) with the determined CA and the calculated Ee(max) the Cfree was calculated based on Cfree ) CD ) CA/Ee(max). EXPERIMENTAL SECTION Reagents and Materials. The five studied CP standards shown in Table 1 were purchased from Dr. Ehrenstorfer GmbH. n-Undecane was obtained from Sigma-Aldrich Chemie GmbH. HPLC-grade methanol and acetonitrile were purchased from Merck KGaA. Humic acid was purchased from Fluka (Lot code 45729, Fluka Chemie GmbH, CH-9471 Buchs, Switzerland, SigmaAldrich, Pf, D-89552 Steinheim, Germany). All other chemicals were of analytical grade or above (Merck), and ultrapure water purified by a Milli-Q Gradient system (Millipore, Bedford, MA) was used throughout. Individual standard stock solutions of chlorophenols (500 mg/ L) were prepared by dissolving 10 mg of standards in 20 mL of HPLC-grade methanol. A working solution (5 mg/L) containing 4802

Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

the five target analytes was prepared by appropriate dilution of the stock solutions with water. The standard stock solutions and working solution were stored at 4 °C. Acceptor buffer solution (1.0 M NaHCO3, pH 9.60) was prepared by dissolving appropriate NaHCO3 in water and adjusting the pH by adding 50% sodium hydroxide. Donor phosphate buffer solution was prepared by dissolving an appropriate amount of NaH2PO4 in water and adjusting to expected pH value by dropwise adding 50% sodium hydroxide. The 50/280 Accurel PP polypropylene hollow fiber tubing (50µm wall thickness, 280-µm inner diameter, 0.1-µm pore size) was obtained from Membrana GmbH (Wuppertal, Germany). BD Micro-Fine Syringe (with a needle of 0.30-mm outer diameter and 8-mm length, 0.5 mL, prepared for U-100 insulin injection), obtained from BD Consumer Healthcare, was used to fill the acceptor into the lumen of the hollow fiber for extraction and to flush out the acceptor into a small glass vial (50 µL, Alltech) after extraction. Instrument. The HPLC equipment used included a 515 HPLC pump (Waters, Milford, MA) and a Waters 996 photodiode array detector. A personal computer equipped with a Millenium32 Chromatography Manager (Waters) was used to process chromatographic data. A Lichrospher 100 RP-C18 endcapped column (250 × 4.0 mm, particle size 5 µm) was used as the analytical column. A pH 211 microprocessor pH meter (Hanna Instruments) used to adjust the donor and the acceptor buffer pH and to measure the sample pH. A B-212 pH meter (Horiba) or the pH 211 microprocessor pH meter equipped with an Orion 98-10 micropH electrode (Thermo Electron Co.), which can measure the pH of a few microliters of solution with precision of (0.02 pH unit, was adopted to detect the acceptor pH after extraction. Extraction Procedure. The hollow fiber supported liquid membrane sampling device shown in Figure 1 was prepared as follows: Hollow fiber pieces, of ∼15 cm length, were connected to the needle of the BD Micro-Fine Syringe holding ∼0.5 mL of the acceptor solution. Then the plunger of the syringe was depressed to flush out ∼0.1 mL of acceptor to wash and fill the lumen of the hollow fiber. The fiber was dipped into the organic solvent (undecane) for a few seconds to impregnate the pores of the hollow fiber wall, thus forming the organic liquid membrane. After that, the lumen of the fiber was flushed slowly with the rest (∼0.4 mL) of the acceptor solution, at the same time completely filling the lumen with acceptor without any air bubbles. To seal the fiber, the two ends were folded three times, enveloped with a

Figure 1. Schematic diagram of preparing the extraction devices, sampling water samples, and collecting extract. (A) Filling the lumen of a hollow fiber with acceptor buffer; (B) dipping the hollow fiber into organic solvent for forming the liquid membrane; (C), sealing the two folded ends of hollow fiber with aluminum foil; (D) prepared hollow fiber supported liquid membrane sampling device; (E), sampling water samples; (F), collecting the acceptor buffer solution into a small vial.

strip of aluminum foil, and inserted into a piece of small glass tubing. After this preparation, the obtained sampling device has an effective fiber length of ∼11 cm with sampling phase volume of ∼7 µL. The whole sealed fiber, with filled acceptor and impregnated organic solvent, was immersed into water and shaken ∼1 min to wash out surplus organic solvent. After this, the hollow fiber sampling device was ready for sampling. For sampling, the hollow fiber sampling device was immersed into the ∼1050 mL sample solution held in a 1000-mL capped volume flask with zero headspace. After static sampling in dark for the selected time, the hollow fiber sampling device was harvested and the water on the fiber surface was absorbed by a paper tissue. One of the sealed ends of the fiber was cut, and the fiber was connected to the needle of a BD Micro-Fine Syringe filled with air, then the other sealed end was cut, and the acceptor with extracted analytes was flushed into a clean and dried 50-µL glass vial. Normally 6-7 µL of acceptor could be collected, of which 5 µL was manually aspirated into a HPLC microsyringe and injected into the HPLC system for analysis. Before aspiration, the HPLC microsyringe was sequentially washed 3 times with water, acetonitrile, and water, respectively. Samples can also be injected into the HPLC system by an autosampler. HPLC Separation. HPLC separation of chlorophenols was conducted by using a mixture of acetonitrile and phosphate buffer

(60:40, v/v) as mobile phase at a flow rate of 1.0 mL/min. To prepare the phosphate buffer, 0.1 mol of sodium dihydrogen phosphate was dissolved in ∼900 mL of water, and the solution was adjusted to pH 2.5 by dropwise addition of phosphoric acid. After this, 50 mL of glacial acetic acid was added, and the solution was diluted to 1000 mL with water. The five chlorophenols were baseline separated in 14 min, and the peak area difference between chlorophenols prepared in water and prepared in 1 mol/L sodium carbonate buffer was less than 10%. Sample Collection. River water was collected from Ho¨je River in the south suburb of Lund, Sweden. Leachate water was collected from Kristianstad, Sweden. The dissolved organic carbon (DOC, mg/L), particles (mg/L), and carbon content of the particles (% C) were 13.4, 2.0, and 11.9 for the river water; and 125, 170, and 8.2 for the leachate water, respectively. Filtrate of leachate water was obtained by filtering the leachate water with a 0.8-µm membrane. For measuring the Cfree in sterilized sample solution, 100 mg/L HgCl2 17 was added to field samples prior to spiking with CPs in order to avoid biodegradation. Water samples were sampled with the hollow fiber supported liquid membrane device without any other further pretreatment. (17) OECD Guideline for Testing of Chemicals. Inherent Biodegrad ability. Concawe Test; OECD Environmental Health and Safety Publication, 2001.


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Figure 2. Effect of agitation (shaking, stirring or static) on the time required to reach equilibrium. Donor: 500 mL of 50 mM KH2PO4 buffer (pH 7.00) spiked with 50 µg/L CPs. Acceptor: 50 mM NaHCO3 buffer (pH 10.00). Sample solutions were sampled by shaking (250 rpm), slow stirring or static with a headspace of ∼60 mL. (a) Static sampling of CP, DCP, TrCP, TeCP, and PCP; (b) static, stirring, or shaking sampling of TrCP.

Data Processing. All experiments were conducted at least twice, and the mean values were reported. Some of the experimental values (Figures 2 and 3) were also fitted (using GraphPad Prism, ver. 4.1, GraphPad Software, inc, San Diego, CA) to a firstorder one-compartment uptake model modified from ref 9:

Ee(t) ) D(1 - e-kt) where k is the rate constant and D is the distribution coefficient that is equivalent to Ee at equilibrium. This model can also be adopted to estimate equilibrium sampling times (e.g., t95% ) ln(0.05)/(-k)), though a more accurate model that relates sampler construction (membrane thickness, surfaces area, volume, etc.) to equilibration time was reported.18 RESULTS AND DISCUSSION Primary Optimization of SLM Parameters. Primary experiments were performed to select the dimension of hollow fiber, organic solvent used as liquid membrane, and buffer species used as donor and acceptor. Two kinds of polypropylene hollow fibers with dimensions of 280/50, and 240/29 (inner diameter/wall thickness, µm) were tested in this study. Although the latter required a shorter time to reach equilibrium, it is not as convenient to handle due to its thin wall. Therefore, the polypropylene hollow fiber with an inner diameter of 280 µm and a wall thickness of 50 µm was adopted in this study. In other studies, the most commonly used hollow fibers for supporting organic solvent are polypropylene capillary membranes with a wall thickness of 200 (18) Divine, C. E.; McCray, J. M. Environ. Sci. Technol. 2004, 38, 1849-1857.


Figure 3. Effect of sample pH on the equilibrium time. Donor: 500 mL of 50 mM KH2PO4 buffer (pH 5.00 or 8.00) spiked with 50 µg/L CPs. Acceptor: 50 mM NaHCO3 buffer (pH 10.00). (a) pH 8.00; (b) pH 5.00 (inset: expanded Ee scale).

µm and internal diameters in the range of 600-1200 µm.12,19 These dimensions are not quite suitable for equilibrium sampling. Large inner diameter results in a low ratio of acceptor volume to surface area of fiber, leading to longer time to reach the equilibrium of extraction. Additionally, the relatively thick fiber wall (200 µm) will form a thick liquid membrane, which not only makes the analytes spend a long time to transport through the liquid membrane to reach equilibrium but also retains a larger amount of analytes in the liquid membrane and thus needs much larger sample volume to prevent depletion. Therefore, thin hollow fibers with small inner diameters and wall thickness is preferred for ESTM. Undecane, dihexyl ether, and 1-octanol, the three most commonly used organic membrane liquids, were tested in this study. It was found that when 1-octanol was used as liquid membrane, many air bubbles formed on the outer surface of the hollow fiber, and the rate of uptake was much lower compared to the other two organic solvents. Although dihexyl ether provided much higher rate of uptake than undecane, undecane was adopted since former study showed that it gave rise to better selectivity than dihexyl ether.20 Phosphate buffer that can provide the environmentally relevant pH range of 5-8 was adopted as the donor buffer. Sodium bicarbonate, sodium tetraborate, glycine, and phosphate buffers, the commonly used buffer systems for a pH higher than 9, which is needed for efficiently trapping the CPs, were tested in this study. Table 2 shows the acceptor pH after 72-h sampling of different samples including river water, leachate water, and phosphate buffer. As can be seen, for all the studied samples, carbonate buffer (19) Psillakis, E.; Kalogerakis, N. Trends Anal. Chem. 2003, 22, 565-574. (20) Knutsson, M.; Mathiasson, L.; Jo¨nsson, J. Å. Chromatographia 1996, 42, 165-170.

Table 2. Acceptor Buffer pH after Sampling 72 h with Different Buffer Solutions as Acceptor acceptor buffer (initial conditions)

sample phosphate buffer (30 mM, pH 7.0) river water (pH 7.6) leachate water (pH 7.3)

sodium carbonate (1.0 M, pH 9.6)

glycine (1.0 M pH 9.6)

sodium tetraborate (0.10 M, pH 9.6)

sodium phosphate (1.0 M, pH 11.5)

10.0

9.1

9.4

10.0

9.5

8.9

8.7

9.5

8.8

8.6

8.1

8.3

gave the highest pH values after sampling, which means that carbonate buffer provided the highest buffer capacity. It is interesting that, after sampling the phosphate buffer sample for 72 h, the carbonate acceptor pH increased from 9.6 to 10.0. The probable cause for this unusual behavior is that the H2CO3 (CO2) concentration in ultrapure water, used for preparing the CP standard sample solutions, is very low and the H2CO3 (CO2) in the acceptor will be extracted into the sample solution, increasing the pH of the acceptor. Sodium bicarbonate buffer was selected as acceptor in the following studies. Time Required To Reach Equilibrium. To prevent biasedlow measurements in equilibrium sampling, it is very important to maintain the sampling until the sampler and its surroundings have reached a thermodynamic equilibrium.9 Thus, parameters that can influence the time to reach equilibrium were investigated in detail. Typically, the enrichment factor Ee increases with time to a constant value Ee(max). As criteria that a thermodynamic equilibrium is attained, the following are required: (1) the increase of Ee has almost stopped and the concentration of the extractable form of a analyte (nondissociated form of a weak acid) in the acceptor reaching 90% of that in sample (donor); (2) Ee is close to the relevant calculated D. It is well known that agitation such as shaking and stirring can decrease the equilibration time. By using 500 mL of 50 mM KH2PO4 buffer (pH 7.00) spiked with 50 µg/L CPs as donor, and 50 mM NaHCO3 buffer (pH 10.00) as acceptor, sampling with shaking (250 rpm), slow stirring and static sample solutions were compared. Since efficient shaking requires an appropriate headspace, these experiments were conducted with a headspace of ∼60 mL. Results shown in Figure 2 indicate that, for all the studied compounds, the time required to reach equilibrium with agitation was about half of that without agitation, and the time required to reach equilibrium increased with increasing enrichment factor; i.e., CP takes 8 h while PCP takes more than 24 h to reach equilibrium in static sampling. Figure 2 also demonstrates that, with shaking, the Ee value increased with sampling time to a maximum value and then decreased gradually. This is probably because the liquid membrane organic solvent left the pores of the hollow fiber and formed a microemulsion in the donor and acceptor solutions, and breakthrough through the liquid membrane was gradually established. The breakthrough of liquid membrane resulted in the decrease of acceptor pH and thus decrease of the Ee value. Further support for the microemulsion formation hypothesis is that, for all the CPs and especially for the more hydrophobic compounds TeCP and PCP, the maximum

Ee value with shaking is always larger than that of static sampling. This result suggests that some analytes were enriched in undecane microdrops scattered into the acceptor due to microemulsion formation and were determined in the following HPLC analysis. It was reported that octanol can form microemulsions in the water phase, thus preventing the reliable determination of Kow values with the shake-flask procedure for compounds with large Kow values, and the “slow-stirring” method is recommended.21 In conclusion, strong shaking should be avoided in order to circumvent the formation of microemulsion of the liquid membrane solvent in the sample and acceptor solutions. Slow stirring can efficiently reduce the equilibrium time and thus can be adopted in equilibrium sampling. Since the purpose of this study is to develop a passive in situ sampling procedure that can be applied for field sampling, the static procedure was adopted as a conservative choice in the following studies. When field sampling is performed for surface water, the equilibration time should be much shorter as a wave or flow of water has the same function as slow stirring. The influence of donor buffer concentration was studied using 50 mM NaHCO3 buffer (pH 10.00) as acceptor and 500 mL of 10, 50, or 200 mM KH2PO4 buffer (pH 7.00) spiked with 50 µg/L CPs as donor solutions. Results (see Supporting Information) demonstrated that donor buffer concentration has no significant influence on the time required to reach equilibrium, and equilibrium was reached in 24 h for all these studied CPs except that it took ∼48 h to reach equilibrium for PCP in 10 mM KH2PO4 buffer. With 500 mL of 50 mM KH2PO4 buffer (pH 7.00) spiked with 50 µg/L CPs as donor, the effect of acceptor buffer concentration was studied by using 50, 500, and 1000 mM NaHCO3 buffer (pH 10.00) solutions as acceptor, respectively. Results (see Supporting Information) showed that the influence of acceptor buffer concentration was analyte dependent. For DCP and TrCP, the equilibration time increased with the acceptor buffer concentration. For TeCP and PCP, however, the shortest equilibrium time was obtained when 1000 mM NaHCO3 buffer was used as acceptor while the 500 mM NaHCO3 buffer required the longest time to reach equilibrium. The effect of sample salinity was studied by comparing the time required to reach equilibrium with and without 500 mM of sodium chloride (∼3% NaCl) in the donor buffer solutions. Results (see Supporting Information) showed that the salinity in the sample solution did not influence the time required to reach equilibrium. But the Ee value increased with the presence of 500 mM of sodium chloride in the donor solution, which is supposed to be the result of a salting-out effect influencing KD. Experiments were also performed to test whether the sample concentration influences the time required to reach equilibrium, and results (see Supporting Information) showed no difference comparing 5 and 50 µg/L CPs. Figure 3 shows the time required to reach equilibrium when sample solutions of pH 5.00 and pH 8.00 were sampled, respectively. For CP, equilibrium was reached in 4 h at both sample pH values. This is because its pKa value (9.2) is very close to the acceptor pH value (10.00) that resulted in low D values (∼7) at both pH 5.00 and pH 8.00. For the other four CPs, it takes 24 h (21) De Bruijin, J.; Busser, F.; Seinen, W.; Hermmens, J. Environ. Toxicol. Chem. 1989, 8, 499-512.


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Figure 4. Sample volume for avoiding depletion. Donor: 50 mM KH2PO4 buffer (pH 6.00) spiked with 50 µg/L CPs. Acceptor: 50 mM NaHCO3 buffer (pH 9.60). Sampling time, 72 h.

to reach sampling equilibrium at pH 8.00, which is because their D values are similar (in the range of 76-100). When the sample pH was changed to pH 5.00, however, DCP and TrCP could reach equilibrium in ∼8 h while equilibrium is far from being attained for TeCP and PCP even after 48-h sampling (see inset). This is because DCP and TrCP have relatively low D values (315 and 1244) while TeCP and PCP possess very large D values (approximately 24 000 and 56 000) at this sampling pH. Further experiments showed that if the acceptor was set at pH 9.60, sampling equilibrium could be reached in 72 h for sample solutions with pHs in the range of 6-8, which is the pH range for most environmental water samples. For samples with pH values lower than 6, either sampling time longer than 72 h or acceptor pH lower than 9.60 should be adopted to ensure equilibrium sampling. In the following studies, acceptor pH 9.60 and sampling time of 72 h were adopted. Sample Volume for Avoiding Sample Depletion. In equilibrium sampling, a large enough sample volume should be adopted to avoid sample depletion. For PCP, which possesses the largest D value of 3688 (at pHA ) 9.6 and pHD ) 6.0), the required sample volume calculated according to ref 9 was ∼500 mL. However, this calculation did not consider the amount of analyte enriched and remaining in the organic membrane liquid. Larger sample volumes might be needed when the analytes remaining in the liquid membrane are considered. Figure 4 shows the influence of sample volume on the Ee(max) value, which indicates that 1000 mL of sample is needed to avoid depletion, and this volume was adopted in the following studies. Acceptor Concentration for Tolerating Interferences. In SLM of weak acids or bases, coexisting interferences with acid/ base characteristics similar to the target compounds will be cotrapped in the acceptor. This might change the pH of the acceptor and thus decrease the enrichment factor if large amounts of interferences were trapped and the buffer capacity is not high enough. Hydrazoic acid (HN3) is an inorganic weak acid with pKa ) 4.6 22 and a large absorbance at the wavelength 280 nm, without any binding or reaction with CPs. This was adopted as a model interference to optimize the acceptor buffer concentration regarding tolerance to interferences. The interference study was conducted by sampling 50 µg/L CPs in KH2PO4 buffer (50 mM, pH 6.00) containing 50 mg/L (0.77 mM) NaN3 with various concentrations of acceptor buffer. Results show that the peak area (22) CRC Handbook of Chemistry and Physics, 82nd ed.; CRC Press: Boca Raton, FL, 2001; pp 8-44.


Figure 5. Effect of environmentally relevant sample salinity on Ee(max)/D. Donor: 10 mM KH2PO4 buffer containing 0-500 mM NaCl (pH 7.00) spiked with 100 µg/L CP amd 50 µg/L DCP, TrCP, TeCP, and PCP. Acceptor: 1000 mM NaHCO3 buffer (pH 9.60). Sampling time, 72 h.

deviation resulted from the addition of NaN3 in donor solution decreased with the increasing of NaHCO3 acceptor concentration in the range of 10-1000 mM, and 1000 mM NaHCO3 can tolerate the interference of 100 mg/L NaN3 in the sample solution with a deviation less than 10%. Experiments showed that NaN3 was enriched ∼200-fold in the acceptor after sampling 0.77 mM NaN3; i.e., the NaN3 concentration in acceptor was 150 mM. This high concentration of interfering agent in the acceptor would change the acceptor pH if the acceptor buffer did not possess enough high buffering capacity. Consequently, 1000 mM NaHCO3 (pH 9.60) buffer solution, the highest concentration that can be prepared, was used as acceptor in the following studies. In real samples, however, there might be various interferences that can be trapped by the acceptor; thus, the acceptor pH might change even when as high as 1000 mM NaHCO3 buffer is adopted as acceptor. Table 2 indicates clearly that the NaHCO3 buffer acceptor pH decreased from 9.6 to 9.5 and 8.8, respectively, when river water and leachate water samples were sampled. Therefore, it is necessary to measure the acceptor pH at sampling equilibrium and then calculate the D value based on the measured acceptor and sample pH values. Determination of Equilibrium Enrichment Factor. Real environmental water samples are complex with different pH, salinity, and buffer capacity, which can influence the equilibrium enrichment factor (Ee(max)). By using NaHCO3 buffer solution (1000 mM, pH 9.60) as acceptor, the effect of the variation of these parameters was studied in detail. Figure 5 shows the effect of environmentally relevant salinity (0-500 mM) on the Ee(max)/D ratio. This was studied by sampling a series of sample solutions prepared by adding various amounts of NaCl in 10 mM NaH2PO4 buffer and then adjusting to pH 7.00. Figure 5 demonstrates that the effect of salinity on the enrichment factor is negligible. For each of the studied CPs, the deviation between the highest and the lowest Ee(max)/D ratio was less than 15%. Thus, it is unnecessary to calibrate for the influence of sample salinity. The effects of sample buffer capacity on the Ee(max)/D ratios are shown in Figure 6, which indicates that the buffer capacity has minor influence, and the deviation of Ee(max)/D in the whole environmentally relevant range of buffer capacity (5-200 mM) is within 15% of that obtained using 30 mM NaH2PO4 buffer. Therefore, 30 mM NaH2PO4 buffer was adopted and no calibration for buffer capacity was performed.

Table 3. Determination of the Ee(max)/D Ratios by Sampling Standard Sample Solutions (30 mM NaH2PO4 Buffer Spiked with 50 µg/L CPs (100 µg/L CP)) in the Range of pH 6.00-8.60 with Carbonate Buffer (1000 mM, pH 9.60) as Acceptor sample pH BSa 6.00 6.50 7.00 7.50 8.00 8.60

acceptor pH ASb

BS

AS

6.00 6.50 7.00 7.50 8.00 8.60

9.60 9.60 9.60 9.60 9.60 9.60

9.75 9.86 9.99 10.18 10.35 10.50

mean ( s a

Before sampling.

b

Ee(max)/D CP

DCP

TrCP

TeCP

PCP

0.60 0.75 0.78 0.88 0.93 1.09

1.61 1.54 1.71 1.56 1.41 1.40

1.16 1.17 1.27 1.25 1.18 1.31

1.15 1.21 1.19 1.14 1.03 1.04

0.90 1.00 1.01 0.92 0.86 0.85

0.84 ( 0.17

1.54 ( 0.12

1.22 ( 0.06

1.13 ( 0.08

0.92 ( 0.07

After sampling

Figure 6. Effect of sample buffer capacity on Ee(max)/D. Donor: 5-200 mM KH2PO4 buffer (pH 7.00) spiked with 100 µg/L CP and 50 µg/L DCP, TrCP, TeCP, and PCP. Acceptor: 1000 mM NaHCO3 buffer (pH 9.60). Sampling time, 72 h.

The effect of sample pH was studied by spiking 100 µg/L CP and 50 µg/L DCP, TrCP, TeCP, and PCP in NaH2PO4 buffer (30 mM, various pH). Results shown in Table 3 indicate that after sampling for 72 h the acceptor pH did increase significantly while the sample pH did not change. Table 3 also indicates that almost constant Ee(max)/D ratios were obtained under different sample pH, and the deviation of Ee(max)/D in the studied pH range (68.6) is within 15%. The Ee(max)/D ratios of all the CPs were all ∼1. Some CPs showed larger differences from 1, probably because the calculation of the D value strongly depends on the pKa value adopted. Since various pKa values are reported in the literature, various values of D will be obtained. For instance, if pKa ) 6.4 23 was adopted for DCP, the calculated Ee(max)/D ratio at sample pH 7.00 becomes 0.52 instead of 1.71 based on pKa ) 7.5. The Ee(max)/D values shown in Figures 5 and 6 and Table 3, which were determined on different days, demonstrated their relatively good reproducibility (RSD < 15%). The deviation of Ee(max)/D values probably come from mainly the measurement of equilibrium acceptor pH. The influence of Ee(max)/D deviation can be reduced by determining the Ee(max)/D ratio as a kind of external calibration in each batch of experiments. According to the above investigation, we can conclude that, with the measured sample and acceptor pH values at equilibrium, (23) Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed.; John Wiley and Sons: New York, 1978; Vol. 5, p 864. (24) Schellenberg, K.; Leuenberger, C.; Schwarzenbach, R. P. Environ. Sci. Technol. 1984, 18, 652-657. (25) Smith, S.; Furay, V. J.; Layiwola, P. J.; Menezes-Filho, J. A. Chemosphere 1994, 28, 825-836.

the equilibrium enrichment factor (Ee(max)) of each CP could be obtained from D, calculated based on eq 7, and the Ee(max)/D ratio determined in each batch of experiment. Analytical Performance. As described above, the linear range and detection limit of the proposed sampling procedure depended on the sample pH. In this present study, these characteristics were determined at sample pH 7.00 by sampling five standard sample solutions (prepared in 30 mM NaH2PO4 buffer) covering the concentration range of 1-100 µg/L CPs (2-200 µg/L of CP). Results shown in Table 4 indicate this procedure possesses excellent linearity (R2 ) 0.9934-0.9993), good precision at the 5 µg/L level (RSD ) 2-12%, n ) 3), and low detection limits (0.1-5 µg/L). Obviously, these detection limits are only valid for standards prepared with reagent water. Detection limits could be higher for real water samples. Interfering compounds could influence the detection, but also, changes of the final pH of the acceptor could lead to lower enrichment factors. The second effect could be counteracted by using higher initial acceptor pH. Effect of Humic Acid on Free Concentration. Synthetic sample solutions (pH 7.00) containing 30 mM NaH2PO4 and certain concentrations of humic acid were used to study the effect of humic acid on the Cfree of CPs. Before sampling, these solutions were spiked with 100 µg/L CP, and 50 µg/L DCP, TrCP, TeCP, and PCP and stirred (with zero headspace) for 1 h to ensure equilibrium between CPs and humic acid. After sampling for 72 h, the acceptor was collected and analyzed by HPLC to obtain the CA values based on calibration curves obtained by direct injection of standard CPs prepared in 1000 mM NaHCO3 solution. The enrichment factor Ee(max) was calculated based on measured sample and acceptor pH values and the determined Ee(max)/D ratios, and the free concentrations of CPs were calculated with the obtained Ee(max) and CA values based on eq 7. Results shown in Table 5 indicate that the Cfree decreased with the increase of added humic acid concentration, and in 50 mg/L humic acid solution (∼17 mg/L DOC), only ∼60% of the total concentration is presented as freely dissolved concentration. A similar strong interaction between humic acid and polar organic compounds was reported earlier and attributed to the electrostatic interaction by Lutzhoft et al.26 Application. The proposed procedure was applied to determine the free concentrations of CPs in river water and leachate (26) Lutzhoft, H.-C. H.; Vaes, W. H. J.; Freidig, A. P.; Halling-Sorensen, B.; Hermens, J. L. M. Environ. Sci. Technol. 2000, 34, 4989-4994.


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Table 4. Analytical Performance of the Proposed Sampling Procedure

a

compd

linear range (µg/L)

regression eq of peak area (y) to sample concn (x, µg/L)

correl coeff (R2)

precision (RSD %, n ) 3)a

detection limit (µg/L)

CP DCP TrCP TeCP PCP

2-200 1-100 1-100 1-100 1-100

y ) 19.39x - 43.59 y ) 1246x - 136.7 y ) 1124x - 796.1 y ) 1196x - 1432 y ) 576.4x - 1936

0.9985 0.9993 0.9984 0.9988 0.9934

2 8 7 12 12

5 0.1 0.1 0.2 2

Determined at 10 µg/L CP, 5 µg/L DCP, TrCP, TeCP, and PCP level.

Table 5. Determination of Free Concentration (Cfree) of Chlorophenols in Synthetic Water Samples by the Proposed Methoda compd CP

DCP

TrCP

TeCP

PCP

humic acid concn (mg/L)

Cfree (µg/L, mean ( s, n ) 2)

0 5 10 50 0 5 10 50 0 5 10 50 0 5 10 50 0 5 10 50

99 ( 2 70 ( 12 58 ( 8 40 ( 2 52 ( 1 40 ( 6 38 ( 1 31 ( 5 49 ( 1 39 ( 5 36 ( 2 29 ( 5 50 ( 4 42 ( 6 41 ( 3 33 ( 3 51 ( 8 40 ( 4 41 ( 2 29 ( 3

a Samples (pH 7.00) prepared by spiking 100 µg/L CP, and 50 µg/L DCP, IrCP, TeCP, and PCP and various concentrations of humic acid in 30 mM NaH2PO4 buffer.

water samples. For both sample types, all CPs were under the detection limits. Therefore, free concentrations were determined after spiking with CPs standards. To confirm the applicability of the proposed method for measuring free concentration, two parallel sets of samples were at first sterilized with 100 mg/L HgCl2 and then spiked with CPs standards. After that, one set of samples was sampled during the 72 h after spiking, while the other set of samples was sampled during 96-168 h after spiking. Results shown in Table 6 demonstrated that there were no significant differences between the free concentrations obtained by these two sampling times; thus, there was no degradation during sampling and the determined free concentrations were reliable. As shown in Table 6, there is significant difference between the free concentrations in leachate water and the filtrate of leachate water when the same amounts of CPs were spiked. This result indicates that parts of the spiked CPs were sorbed to the particles present in leachate water. What should be noted is that, although the samples were very dirty, the obtained chromatograms (see Supporting Information) were as clean as those from the standard solutions, which 4808


demonstrates the high selectivity of the proposed sampling procedure. CONCLUSIONS A novel ESTM procedure based on hollow fiber supported liquid membrane extraction was proposed for equilibrium sampling of freely dissolved concentrations of chlorophenols in environmental water samples. The developed disposable sampling device, made from a single polypropylene hollow fiber, is very inexpensive and easy to prepare. The freely dissolved concentration of five chlorophenols, with a wide range of pKa (4.9-9.2) and log Kow (2.35-5.01), in river water and leachate water samples, were successfully determined at a microgram per liter level with high selectivity. Since a few microliters of acceptor buffer solution in a hollow fiber were deployed in 1000 mL of samples to avoid sample depletion and the sampling time was 72 h to reach equilibrium, buffer solutions with high buffer capacity were preferred to be adopted as acceptor. It was demonstrated that carbonate buffer possesses the highest buffer capacity in the pH range of 9.611.5. As analytes and interferences were enriched over 1000-fold, the acceptor pH value might change after sampling even when 1000 mM carbonate buffer was used as acceptor. Therefore, it is very important to measure the acceptor and sample pH values at equilibrium to calculate distribution coefficients and thus the free concentration. High-precision measurement of acceptor and sample pH are required as D is exponentially related to pH. Basically, Ee(max) agreed well with D at equilibrium to an extent that depends on the pKa value adopted to calculate D. Environmentally relevant sample buffer capacity, salinity, and pH have minor influence on the Ee(max)/D ratio. For higher accurate determination of Cfree, it is recommended to determine the Ee(max)/D ratio in every analysis batch. The equilibrium sampling time depends on the D value and can be reduced efficiently by slow stirring the samples if sampling is conducted in the laboratory. For field sampling, thinner hollow fibers should efficiently reduce the equilibrium time. Though small acceptor volume (∼7 µL) was adopted in this study, larger acceptor volumes, up to 30 µL can be obtained by simply using a 50-cm-long hollow fiber. Principally, this proposed fiber sampling device is suitable for sensing the Cfree of all analytes that can be extracted with the SLM technique (typically weak acids or bases, as well as many metal ions) by limiting the “manipulation” in the extraction procedure to the membrane and the acceptor solution while adopting a sample volume that is sufficiently large to ensure negligibledepletion extraction.

Table 6. Free Concentration (Cfree) of Chlorophenols (µg/L, Mean of 3 Measurements) in Water Samples after Spiking with Standards sample without sterilizing

sample sterilized with 100 mg/L HgCl2 Cfreea

Cfree sample river water CP DCP TrCP TeCP PCP leachate water CP DCP TrCP TeCP PCP filtrate of leachate water CP DCP TrCP TeCP PCP a

spiked

dissoc

100 50 50 50 50

ndb 6.7 8.2 9.1 8.8

200 100 100 100 100

1 11 19 28 9.4

200 100 100 100 100

2 11 24 37 50

nondissoc

total

spiked

dissoc

nondissoc

total

nd 5.3 1.6 0.072 0.018

nd 12 9.8 9.2 8.8

400 200 200 200 200

6 (7) 15 (11) 58 (56) 50 (52) 87 (106)

299 (354) 14 (12) 14 (14) 0.50 (0.52) 0.22 (0.26)

305 (361) 29 (23) 72 (70) 50 (53) 87 (106)

87 23 9.7 0.56 0.047

88 34 29 29 9.5

800 400 400 400 400

5 (6) 43 (43) 95 (98) 155 (169) 141 (166)

397 (463) 68 (68) 38 (39) 2.4 (2.7) 0.56 (0.66)

402 (469) 111 (111) 133 (137) 157 (172) 142 (167)

200 100 100 100 100

2 (2) 12 (9) 24 (22) 38 (41) 44 (39)

153 (177) 19 (15) 9.7 (8.8) 0.61 (0.65) 0.17 (0.15)

155 (179) 31 (24) 34 (31) 39 (42) 44 (39)

178 21 11 0.74 0.25

180 32 35 38 50

Data in parentheses are obtained by sampling samples during 96-168 h after spiking of standards. b nd, not detected.

Perspectives for Field Sampling. Most sampling and extraction techniques that are used in the laboratory can hardly be applied in the field, since they require a defined sample volume or highly controlled and standardized sampling conditions. ESTM requires only that the acceptor solution is brought into equilibrium with a sufficiently large “sample volume”, which makes it very suited for field sampling. ESTM has in this respect many similarities with other equilibrium sampling devices,9 with sensors and with selective electrodes. All these techniques can be applied in many different situations. Disposable hollow fibers seem an attractive format to be applied in field sampling, since they combine low cost, simplicity, and a high surface-to-volume ratio. This proposed fiber sampling device should be suitable for in situ field sampling, and further testing is being conducted by this research group. ACKNOWLEDGMENT This work was supported by the Foundation for strategic environmental research (MISTRA), Sweden.

NOTE ADDED AFTER ASAP PUBLICATION This paper was inadvertently posted on June 15, 2005, before a correction to the sixth line of the paragraph “Effect of Humic Acid on Free Concentration” was made. The version posted on July 1, 2005, is correct. SUPPORTING INFORMATION AVAILABLE Plots showing the effects of donor buffer concentration, acceptor buffer concentration, sample salinity, and sample concentration on the time required to reach equilibrium, as well as typical chromatograms of water samples, This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 26, 2005. Accepted May 17, 2005. AC0503512


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Equilibrium Sampling through Membranes of ... - ACS Publications

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