Potential‐driven peptide extractions across supported liquid membranes: Investigation of principal operational parameters

Fundamental experiments on electromembrane extraction were performed to increase the basic knowledge about the current and the mass transfer of target peptides and background electrolyte ions. Three peptides (angiotensin 2, bradykinin, and enkephalin) were extracted from 500 μL aqueous donor solution (1 mM HCl, positive electrode), through a 200 μm supported liquid membrane (SLM) of 1‐octanol/di‐isobutylketon/di‐(2‐ethylhexyl) phosphate (55:35:10 w/w/w) sustained in the pores of a porous hollow fiber, and into 25 μL aqueous acceptor solution (50 mM HCl, negative electrode) present inside the lumen of the fiber by the application of an electrical potential (50 V) and agitation (1050 rpm). Recoveries were typically in the range of 55–65% after 5 min of extraction and were principally determined by the chemical composition of the SLM and by the applied voltage. The electrical current in the system was measured during the extraction and was close to 350 μA. The current arose to some extent from mass transfer of the target peptides, but the major contribution was due to a background current from di‐(2‐ethylhexyl) phosphate in the SLM and from mass transfer of background electrolytes. Operation at relatively low background current was important to maintain a stable system.     

Electromembrane extraction of peptides--fundamental studies on the supported liquid membrane

A large screening of different components in the supported liquid membrane (SLM) in electromembrane extraction (EME) was performed to test the extraction efficiency on eight model peptides. Electromembrane extraction from a 500 μL acidified aqueous sample containing the model peptides in the concentration 10 μg/mL was used. Extraction time was 5 min with an electric potential of 10 V and 900 rpm agitation of the sample vial. The samples were extracted through a hollow fiber-based SLM with different compositions of organic solvents and carriers. A small volume of acidified acceptor solution (25 μL) was after extraction analyzed directly, or with some dilution, on CE or HPLC. This article has identified mono- or di-substituted phosphate groups as the prominent group of carrier molecules needed to obtain acceptable recoveries. For the organic solvents, primary alcohols and ketones have shown promise regarding recovery and reproducibility, with some differences in selectivity. A new composition of the SLM, namely 2-octanone and tridecyl phosphate (90:10 w/w) has proved to give higher extraction recoveries and lower standard deviation than SLMs previously reported in the literature.     

Drop-to-drop microextraction across a supported liquid membrane by an electrical field under stagnant conditions

Electromembrane extraction (EME) of basic drugs from 10 μL sample volumes was performed through an organic solvent (2-nitrophenyl octyl ether) immobilized as a supported liquid membrane (SLM) in the pores of a flat polypropylene membrane (25 μm thickness), and into 10 μL 10 mM HCl as the acceptor solution. The driving force for the extractions was 3–20 V d.c. potential sustained over the SLM. The influence of the membrane thickness, extraction time, and voltage was investigated, and a theory for the extraction kinetics is proposed. Pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from pure water samples with recoveries ranging between 33% and 47% after only 5 min of operation under totally stagnant conditions. The extraction system was compatible with human urine and plasma samples and provided very efficient sample pretreatment, as acidic, neutral, and polar substances with no distribution into the organic SLM were not extracted across the membrane. Evaluation was performed for human urine, providing linearity in the range 1–20 μg/mL, and repeatability (RSD) in average within 12%.     

Impedometric monitoring of the behavior of the supported liquid membrane in electromembrane extraction systems: An insight into the origin of optimized experimental parameters

Electromembrane extraction (EME) was carried out using a novel instrumentation capable of impedometric monitoring of the system during the extraction. This instrumentation involves a classical two-electrode assembly fed by two time-resolved potential functions, the first for the extraction of analyte and the second for obtaining the impedance information. The impedometric analysis of the system was achieved by Laplace transformation of the current recorded during the extraction. It has been shown that the obtained impedance information can be converted to very useful knowledge about time dependence of double layer capacitance, kinetics of analyte depletion, total permeability of the SLM and the effect of experimental parameters on system behavior. It has also been shown that the impedance analysis is a powerful tool for the estimation of optimum experimental parameters without determination of analyte in the acceptor phase.     

Fast,selective, and sensitive analysis of low-abundance peptides in human plasma by electromembrane extraction

A totally new concept based on electrokinetic migration was evaluated for the extraction of three biologically active peptides from human plasma. Angiotensin 2, leu-enkephalin, and endomorphin 1 migrated from a diluted human plasma sample (2 mL, positive electrode), through a supported liquid membrane (SLM) of 1-octanol, di-isobutylketon, and di-(2-ethylhexyl) phosphate (DEHP) (55:35:10, w/w/w), and into an acidified acceptor solution (25 μL 50 mM HCl, negative electrode) by the application of an electrical potential (20 V) across the SLM. After only five min of extraction, the acceptor solution was injected and analyzed directly by liquid chromatography. The three peptides were quantified by tandem mass spectrometry, with acceptable linearity ranging from 100.0 to 1000.0 pg mL⁻¹ (r² in the range 0.9736–0.9988), and repeatability (RSD) ranging between 15% and 24% (n = 5), using plasma spiked with the three peptides in 100 pg mL⁻¹ concentration. The estimated detection limits (S/N ratio of 3:1) for angiotensin 2, leu-enkephalin, and endomorphin 1, were 60, 24, and 24 pg mL⁻¹, respectively. With this novel approach based on electromembrane extraction (EME) coupled to LC–MS/MS, endogenous concentrations of the peptides were detected in non-spiked human plasma samples, with a total analysis time less than 50 min. These experimental findings were highly interesting, and showed the opportunities for EME with regard to future peptide extractions.     

Exhaustive extraction of peptides by electromembrane extraction

This fundamental work illustrates for the first time the possibility of exhaustive extraction of peptides using electromembrane extraction (EME) under low system-current conditions (<50 μA). Bradykinin acetate, angiotensin II antipeptide, angiotensin II acetate, neurotensin, angiotensin I trifluoroacetate, and leu-enkephalin were extracted from 600 μL of 25 mM phosphate buffer (pH 3.5), through a supported liquid membrane (SLM) containing di-(2-ethylhexyl)-phosphate (DEHP) dissolved in an organic solvent, and into 600 μL of an acidified aqueous acceptor solution using a thin flat membrane-based EME device. Mass transfer of peptides across the SLM was enhanced by complex formation with the negatively charged DEHP. The composition of the SLM and the extraction voltage were important factors influencing recoveries and current with the EME system. 1-nonanol diluted with 2-decanone (1:1 v/v) containing 15% (v/v) DEHP was selected as a suitable SLM for exhaustive extraction of peptides under low system-current conditions. Interestingly, increasing the SLM volume from 5 to 10 μL was found to be beneficial for stable and efficient EME. The pH of the sample strongly affected the EME process, and pH 3.5 was found to be optimal. The EME efficiency was also dependent on the acceptor solution composition, and the extraction time was found to be an important element for exhaustive extraction. When EME was carried out for 25 min with an extraction voltage of 15 V, the system-current across the SLM was less than 50 μA, and extraction recoveries for the model peptides were in the range of 77–94%, with RSD values less than 10%.     

Rapid isolation of angiotensin peptides from plasma by electromembrane extraction

The present study has for the first time demonstrated the isolation of peptides from human plasma by electromembrane extraction (EME). Angiotensin 1, angiotensin 2, and angiotensin 3 migrated from 500 μL of diluted plasma, through a thin layer of 1-octanol and 8% di-(2-ethylhexyl) phosphate immobilized as a supported liquid membrane (SLM) in the pores of a porous hollow fiber, and into a 25 μL aqueous acceptor solution present inside the lumen of the fiber. The driving force for the extraction was a 15 V potential difference applied across the SLM. After only 10 min of EME, the peptides were isolated from diluted plasma (pH 3) with extraction recoveries between 25 and 43%. After optimization, the extraction system was evaluated using spiked plasma samples of angiotensin 2. The evaluation was performed by liquid chromatography electrospray mass spectrometry, showing linearity of angiotensin 2 in the range 2.5–125.0 ng/mL (r² = 0.989), and repeatability (RSD) between 5.6 and 11.6% (n = 6). The results demonstrate the possibility of isolating angiotensin peptides from plasma in only 10 min, using electromembrane extraction. The experimental findings are therefore promising with regard to future peptide extractions.     

Effects of selected operational parameters on efficacy and selectivity of electromembrane extraction. Chlorophenols as model analytes

Effects of organic solvent type, pH value, and composition of donor/acceptor solution on the efficacy of electromembrane extraction (EME) were examined. For the first time, a comprehensive quantitative study, based also on measurements of electric charge passed through the EME system, was carried out, which demonstrates that apart from the pH value, also the nature of counter‐ions in donor and acceptor solution plays a significant role in the electrically induced transfer of charged analytes across supported liquid membranes (SLMs). The EME transfer of model analytes correlated well with electrophoretic mobilities of inorganic cations, which were added to acceptor solutions during their alkalization with alkali metal hydroxides, and were highest for counter‐cations with highest mobilities. Up to a 53‐fold improvement of extraction efficiency was achieved for EMEs using optimized composition of donor (alkalized with KOH to pH 7) and acceptor (10 mM CsOH, pH 12) solutions. Six chlorophenols (CPs) were selected as model analytes due to the wide range of pH values that are required for their ionization and due to their high environmental relevance; quantitative measurements were carried out by CE with UV detection. Extraction recoveries of the six CPs ranged between 14 and 25% for 5 min EMEs at 150 V and 750 rpm across SLMs impregnated with 1‐ethyl‐2‐nitrobenzene. Calibration curves were strictly linear (r² ≥ 0.999) in 0.01–10 μg/mL range, repeatability values of peak areas were between 0.7 and 5.6% and LODs for standard solutions and environmental samples were better than 5 ng/mL.     

Study of the effect of the membrane composition on ion transfer across a supported liquid membrane

The rate of ion transfer across the supported liquid membrane (SLM) is studied in the rotating diffusion cell (RDC), varying the chemical composition of the SLM from net-cloth supported gel membranes to radiation-grafted polymer membranes. Steady-state current–voltage curves are measured as a function of the rotation rate, and values for the standard rate constant, k⁰, are determined for a series of tetraalkylammonium cations from the analysis of the initial slopes and the diffusion limiting currents. The analysis gives values for k⁰ of the order of 10⁻²–10⁻⁴ cm s⁻¹, which is in rather good agreement with the values found in the literature for this type of the system. As controlled delivery of ionic drugs can be achieved by control of the electric current, whereby the SLM acts as a drug reservoir, the study is extended to the release of the anti-Alzheimer drug Tacrine, where ion-exchange fibers are embedded in the membrane as the drug carrier. Our previous transient experiments are also discussed, and it is suggested that their interpretation is seriously hampered by the non-uniform potential distribution, which brings about high capacitive currents.     

Instability mechanisms of supported liquid membranes for copper (II) ion extraction

The instability of supported liquid membranes (SLMs) for use in copper (II) ion extraction was investigated in this paper. The degradation behavior of these SLMs was monitored in situ by electrochemical impedance spectroscopy (EIS). The electrical properties of a SLM cell can be described by an equivalent circuit, R_s(C_mR_m)Q. The model parameters, membrane resistance (R_m) and membrane capacitance (C_m) can be used to characterize the degradation behavior of SLMs. Experimental data for R_m and C_m indicated that the loss of membrane liquid (ML) during the mass transfer process consists of three stages. Results also suggest that emulsion formation was the dominant instability mechanism for these SLMs. The solubility and osmotic pressure were also shown to not be major factors contributing to the instability although both contributed to the loss of the liquid membrane. The pore size of the polymeric support increased during the first run but remained almost constant in subsequent runs.     

Studies on phenol permeation through supported liquid membranes containing functionalized polyorganosiloxanes

In this study, the functionalized, linear, hydrophobic fluid organosiloxane polymers, namely, methylhydrosiloxane–dimethylsiloxane copolymers supported on a polypropylene microporous flat sheet membrane (Celgard 2502 and 2402) have been tested as supported liquid membranes (SLMs) for phenol recovery from aqueous phases into a 0.1 M NaOH phase. The functionalized polymers include, Me₃SiO[MeSi(OR)O]_x[Me₂SiO]_ySiMe₃ (containing x = 15–18, 25–35 and 50–55 mol% of R, where R is –(CH₂)_nNMe₂ (n = 3 or 4 or 6) or –(CH₂)₂OEt pendent organofunctional groups. The functionalities, R, tested were derived from the commercially available 3-dimethylamino-1-propanol and 2-ethoxyethanol as well as newly synthesized 4-dimethylamino-1-butanol and 6-dimethylamino-1-hexanol which have been made for the purpose of this study.

The study showed that phenol permeation expressed as permeate flux through the membranes increases with the larger number of carbon spacers in the alkyl chain of the aminoalcohol pendent, larger porosity of the polypropylene support films, higher mol% of the methylhydrosiloxane portion functionalized and faster flow rates of both the feed and the receiving phases. Phenol permeation was enhanced significantly when the mol% of the methylhydrosiloxane portion was 50–55 or 25–35 with 6-dimethylamino-1-hexanol functionality supported on Celgard 2502.     

Electromembrane extraction of basic drugs from untreated human plasma and whole blood under physiological pH conditions

The present work describes the first systematic study of electromembrane extraction (EME) from biological matrices under physiological conditions. Six basic drugs with protein binding in the range of 20–97% were extracted from untreated human plasma and whole blood through a supported liquid membrane (SLM) consisting of 1-ethyl-2-nitrobenzene impregnated in the walls of a hollow fiber, and into an acidified aqueous solution inside the lumen of the fiber. The electrical potential difference over the membrane reduced the protein binding of the drugs and transported the free drug fraction over the membrane. Recoveries in the range 25–65% were obtained with 10-min extraction time and an applied voltage of only 10 V over the SLM. Interday precision better than 20% RSD and linearity in the range 0.5–10 μg/mL were obtained for nortriptyline and methadone. Extraction from untreated whole blood was also demonstrated with recoveries in the range 19–51%.     

Supported liquid membrane extraction with single hollow fiber for the analysis of fluoroquinolones from environmental surface water samples

In this work, the simple analytical method for the determination of four fluoroquinolone antibiotics: ciprofloxacin, enrofloxacin, norfloxacin and danofloxacin, in environmental surface water samples is described. Sample pretreatment step was performed by the application of a technique based on supported liquid membrane extraction with the configuration of single hollow fiber (HF-SLM). The HPLC system with diode array detection was used for final analysis of studied analytes. Various parameters affecting the extraction efficiency during HF-SLM enrichment, such as type of membrane diluent, pH of donor (sample) and acceptor phases, as well as an enrichment time and salt content of sample were studied. Using the presented hollow-fiber extraction high recovery (70–80%) was achieved. It gave enrichment factor above 100. The detection limits in surface water samples, for the four target antibiotics, were at range 0.01–0.02 μg/l, when 10 ml samples were processed. The obtained results demonstrate the applicability of presented method for the selective extraction of fluoroquinolones in environmental water samples at ultratrace level. Errors, expressed as relative standard deviation (RSD) were below 8%, for all tested concentration levels.     

Influence of temperature on mass transfer in an incomplete trapping supported liquid membrane extraction of triazole fungicides

The influence of temperature in a supported liquid membrane (SLM) extraction of triazole fungicides was investigated. The mass transfer parameters such as diffusion coefficient, flux and apparent viscosity were determined at temperatures ranging from 5 to 40°C. Increase in temperature led to an increase in diffusion coefficient and flux with a flowing acceptor solution. The apparent viscosity also decreased with an increase in temperature. However, the increase in mass transfer parameters did not result in an overall increase in extraction efficiency with a stagnant or circulation acceptor phase. Stripping of the analytes from the membrane into the acceptor phase as well as the configuration of the extraction unit could have limited the influence of temperature on mass transfer. The partition coefficient of analytes from the acceptor solution to the membrane, K_A, was found to be much higher than that from the donor solution to the membrane K_D, thus triazole compounds preferred to remain in the membrane even with an increased extraction temperature.     

Coupled transport of Ag(I) ions through triethanolamine–cyclohexanone-based supported liquid membranes

Facilitated transport of silver(I) ions in acidic medium, across a supported liquid membrane (SLM) by using triethanolamine (TEA) as carrier, dissolved in cyclohexanone, has been investigated. The parameters studied are HNO₃ concentration variation in the feed, pH of the feed solution, carrier concentration in the membrane phase, silver(I) ions concentration in the feed phase and KCN concentration in the stripping phase. Increase in H⁺ concentration by increasing HNO₃ concentration from 0.5 to 1 M results into an increase in silver ions flux but a decrease in flux has been found beyond 1 M HNO₃ concentration in the feed, providing a maximum flux of 3.21 × 10⁻⁷ mol/m² s at 1 M HNO₃. Increase in TEA concentration inside the membrane enhances flux with its maximum value at 2.25 M TEA. Further increase in the concentration of TEA leads to a decreased rate of transport due to the increase in viscosity of membrane liquid. The optimum conditions for Ag(I) ions transport are 1 M HNO₃ (feed), 2.25 M TEA (membrane) and 1.5 M KCN in the stripping phase. It has been observed that Ag(I) flux across the membrane tends to increase with increase in Ag(I) ions concentration in the feed phase. Applying the studied conditions to silver plating waste solutions, Ag ions have been removed up to 99% in a time interval of 5 h.     

Determination of metallo-cyanides by capillary electrophoresis after concentration on supported liquid membranes

A method for the determination of traces of metallo-cyanide complexes by capillary zone electrophoresis is described. The suitability of preconcentration procedures based on supported liquid membranes in a flow system is investigated. Methyltrioctylammonium chloride in dibutyl ether is used as the active component of the membrane liquid. Due to ion-pairing mechanisms enrichment factors ranging from 50 to 600 can be achieved for cyanide complexes of Fe(II), Fe(III), Ni, Co, Pd, Pt, Cr, Au and Ag. The final capillary electrophoretic separation of the metallo-cyanides is performed off-line with a phosphate-triethanolamine buffer at pH 8.5 as the carrier electrolyte. Its separation selectivity and compatibility with the preconcentration procedure are optimized by addition of hexamethonium bromide, sodium perchlorate and sodium cyanide. Detection limits in the low nmol range can be achieved by direct UV detection at 214 nm. An approach for the analysis of free and labile cyanide is discussed which involves the conversion of these species into the nickel cyanide complex. Applications in the fields of environmental monitoring and industrial process control are possible.     

Electromembrane extraction through a virtually rotating supported liquid membrane

Electromembrane extraction (EME) of model analytes was carried out using a virtually rotating supported liquid membrane (SLM). The virtual (nonmechanical) rotating of the SLM was achieved using a novel electrode assembly including a central electrode immersed inside the lumen of the SLM and five counter electrodes surrounding the SLM. A particular electronic circuit was designed to distribute the potential among five counter electrodes in a rotating pattern. The effect of the experimental parameters on the recovery of the extraction was investigated for verapamil (VPL), trimipramine (TRP), and clomipramine (CLP) as the model analytes and 2‐ethyl hexanol as the SLM solvent. The results showed that the recovery of the extraction is a function of the angular velocity of the virtual rotation. The best results were obtained at an angular velocity of 1.83 RadS^?1 (or a rotation frequency of 0.29 Hz).The optimization of the parameters gave higher recoveries up to 50% greater than those of a conventional EME method. The rotating also allowed the extraction to be carried out at shorter time (15 min) and lower voltage (200 V) with respect to the conventional extraction. The model analytes were successfully extracted from wastewater and human urine samples with recoveries ranging from 38 to 85%. The RSD of the determinations was in the range of 12.6 to 14.8%.     

Extraction of molybdenum by a supported liquid membrane method

This is a report on the extraction of molybdenum(VI) ions using a supported liquid membrane, prepared by dissolving in kerosene, the extractant Alamine 336 (a long-chain tertiary amine) employed as mobile carrier. A flat hydrophobic microporous membrane was utilised as solid support. Appropriate conditions for Mo(VI) extraction through the liquid membrane were obtained from the results of liquid-liquid extraction and stripping partition experiments. The influence of feed solution acidity, the carrier extractant concentration in the organic liquid film and the content of strip agent on the metal flux through membrane were investigated. It was established that maximal extraction of metal is achieved at a pH 2.0 if sulphuric acid is used in the feed solution and at a pH value over 11.0 if Na₂CO₃ is used as strip agent. Moreover, the molybdenum extraction through membrane is enhanced when a 0.02 mol l⁻¹ content of the amine carrier in the organic phase is used. The present paper deals with an equilibrium investigation of the extraction of Mo(VI) by Alamine 336 and its permeation conditions through the liquid membrane, and examines a possible mechanism of extraction.     

In‐line coupling of supported liquid membrane extraction across nanofibrous membrane to capillary electrophoresis for analysis of basic drugs from undiluted body fluids

Planar polyamide 6 nanofibrous membrane was for the first time used in direct coupling of supported liquid membrane (SLM) extraction to CE analysis. Disposable microextraction device with the nanofibrous membrane was preassembled and stored for immediate use. The membrane in the device was impregnated with 1 µL of 1‐ethyl‐2‐nitrobenzene and the device was subsequently filled with 10 µL of acceptor solution (10 mM HCl) and 15 µL of donor solution (sample). The device was in‐line coupled to CE system for selective extraction and direct injection, separation and quantification of model basic drugs (nortriptyline, haloperidol, loperamide and papaverine) from standard saline solutions (150 mM NaCl) and from undiluted human body fluids (urine and blood plasma). Compared to standard polypropylene supporting material, the nanofibrous membrane demonstrated superior characteristics in terms of lower consumption of organic solvents, constant volumes of operational solutions, full transparency and possibility to preassemble the devices. Extraction parameters were better or comparable for the nanofibrous vs. the polypropylene membrane and the hyphenated SLM‐CE method with the nanofibrous membrane was characterized by good repeatability (RSD ≤ 11.3%), linearity (r² ≥ 0.9953; 0.5–20 mg/L), sensitivity (LOD ≤ 0.4 mg/L) and transfer (27–126%) of the basic drugs.     

Parameters affecting electro membrane extraction of basic drugs

Thirty-five different basic drugs were extracted by electro membrane extraction (EME), from acidified samples containing HCl as the BGE, through an organic solvent immobilized in the pores in the wall of a porous hollow fiber (supported liquid membrane, SLM), and into an acidified acceptor solution (HCl) in the lumen of the hollow fiber by the application of an electrical potential difference of 50 V. With 2-nitrophenyl pentyl ether (NPPE) as the SLM, and with 10 mM HCl as BGE in the sample and acceptor solution, singly charged basic drugs with log P >2 were extracted with recoveries in the range 30-81% within 5 min. For doubly charged basic drugs, extraction was effectively enhanced by decreasing the concentration of HCl in the sample from 10 to 0.1 mM, reducing the ionization of the analytes. For medium polar analytes (1 < log P < 2), an ion balance of 0.01 was combined with addition of tris-(2-ethylhexyl) phosphate (TEHP) to the SLM, and this provided recoveries in the range 36-70%. The ion balance was defined as the concentration ratio of BGE between the sample and the acceptor solution. For the most polar drugs (log P <1), EME was accomplished with an ion balance of 0.01 and with di-(2-ethylhexyl) phosphate (DEHP) added to the SLM, but in spite of this, recoveries were in the range of only 4-17%.