Flow Chronopotentiometry with Ion-Selective Membranes for Cation

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Flow Chronopotentiometry with Ion-Selective Membranes for Cation, Anion and Polyion Detection Majid Ghahraman Afshar, Gaston A. Crespo, and Eric Bakker Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00141 • Publication Date (Web): 02 Mar 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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Analytical Chemistry

Flow Chronopotentiometry with Ion-Selective Membranes for Cation, Anion and Polyion Detection Majid Ghahraman Afshar, Gastón A. Crespo, and Eric Bakker*

Department of Inorganic and Analytical Chemistry, University of Geneva, Quai Ernest-Ansermet 30, CH1211 Geneva, Switzerland.

Corresponding Author: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT We report here on the development of a chronopotentiometric readout for ion-selective electrode that allows one to record transition times in continuous flow conditions without the necessity to stop the flow. A sample plug of 150 µL is injected into the carrier solution (0.5 mM NaCl) and subsequently transported to the detection cell (~20 µL) at moderate flow rates (~0.5 mL min-1), where a short current pulse (5s) is applied between the ionophore-based working electrode and a biocompatible and non-polarizable Donnan exclusion anion-exchanger membrane reference/counter electrode. Flow conditions bear an influence on the thickness of the aqueous diffusion layer and result in a shift of the chronopotentiometric transition time with respect to stopped flow. Two models based on rotating disk electrodes and flow chronopotentiometry at metal-based electrodes were used to corroborate the data. The method was successfully applied to the determination of calcium, chloride, alkalinity, acidity and protamine with a range of ion-selective membranes. Because of the limiting exposure time of ca. 20-s of the membranes with the sample, this approach is demonstrated to be useful for the detection of protamine in the therapeutic range of undiluted human blood. Keywords: chronopotentiometry; flow conditions; protamine; human blood; ionophore-based membranes


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INTRODUCTION Controlled current techniques such as chronopotentiometry have recently been applied to perturb ion concentrations in the vicinity of different electrochemical systems. A wide range of materials that include metal electrodes and ionophore-based or ion-exchanger-based membrane electrodes have been employed with the aim of performing fundamental electrochemical studies 1-3, electrodialysis 4-11, and ion sensing. 12 In analogy to early chronopotentiometric experiments at metal electrodes13-15, the applied current translates to an imposed concentration gradient of fixed amplitude at the electrode surface. As mass transport in the sample is commonly the rate limiting step, the established concentration gradient can often not be maintained for the duration of the experiment, and local concentration depletion is eventually observed in the form of a potential change with time. Redox turnover (at metal electrodes) and ion transfer processes (at ion-selective membranes) are assumed to be much faster than ion diffusion in the sample, allowing one to use the Sand equation in order to relate the observed transition time to the concentration in the sample bulk.16 Recently, Abbas et al.17 demonstrated the chronopotentiometric detection of chloride using silver metal electrodes. Two identical silver/silver chloride elements were used as working and reference electrode whereas a platinum rod served as counter electrode. The depletion of chloride ions at the working electrode was visualized as an inflection of the chronopotentiogram (V(WE)-V(RE) vs. t). Consequently, the chloride concentration at the electrode surface was described in terms of the Sand and Nernst equations considering the applied current, the potential difference, the transition time (τ), the geometry of the electrode and the bulk chloride concentration. Note that the use of an Ag/AgCl electrode material may limit the determination of low concentrations of chloride (~10-5 M) and of ions in biological samples due to the lack of biocompatibility.18-19 In a similar direction, our group addressed some unresolved analytical challenges using flash chronopotentiometry at ionophore-based membrane electrodes. For example, i) the direct detection and speciation of free and bound calcium and lipophilic drugs such as phenytoin (i.e., Ca-NTA, phenytoinBSA);20-21 ii) the determination of total and p-alkalinity;22-23 iii) universal, nearly non-discriminative anion and cation detection with Donnan Exclusion Ion-Exchanger Membranes;24 iv) selective ion-detection with ionophore-based membranes backside contacted with lipophilic ferrocene25-26; and v) reversible protamine/heparin detection in undiluted human blood.27-28



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This latter system, which consists of the determination of protamine and heparin in human blood samples, is of high practical relevance. Protamine is an important polycation regularly used to neutralize heparin during a range of surgical procedures (typical range of 20-120 ppm). The reference methodology for heparin detection is activated clotting time (ACT), which is only semi-quantitative since reflects the physiological response to administered heparin. Moreover, ACT varies with time since heparin is continuously metabolized in the body.29-31 As a result of this, there has been continued effort to develop reliable potentiometric sensors for heparin or protamine detection, although a potentiometric readout results in poor sensor reversibility.32-39 More recent, Bell-Vlasov et al.40 has presented a pulstrode PVC ionophore-based membrane electrode for protamine detection originally reported by Gemene et al.

41

but in flow-conditions using a wall-jet cap

mounted on the electrode. This elegant approach improved on two aspects for clinical analysis: a reduced injection volume and increased sampling frequency. The experiments were unfortunately not successful in undiluted human blood

40

and did not give transition times as readout signal. In contrast to transition

times, which give linear calibration curves, the sampling of potential at a fixed time results in sigmoidal response curves in analogy to zero current potentiometry that are more easily influenced by changes in the background electrolyte. Yet, the use of dynamic electrochemistry with ionophore-based membranes under flow conditions is a promising approach and is here adopted for the first time in combination with a flash chronopotentiometry readout.27 The aim of this work was to demonstrate the usefulness of ionophore-based membrane electrode readout with chronopotentiometry in flow conditions. This principle allows one to measure the concentration of the analyte in flow conditions using a reduced amount of sample (in the range of 50 to 120 µL). In the design of the flow cell, an anion exchanger membrane was used as a counter/reference electrode with the aim of performing chronopotentiometric analysis with a 2-electrode system.18 A wide range of ions (calcium, proton, chloride, hydroxide and protamine) was determined in artificial and biological samples. The experimental data were validated with two established theories based on rotating disk electrode (RDE) and flowing stream chronopotentiometry.


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EXPERIMENTAL Materials and Chemicals Tetradodecylammonium chloride (TDDA), 2-nitrophenyl octyl ether (o-NPOE), protamine sulfate salt from herring (P4505), Trizma hydrochloride (Tris·HCl), sodium chloride, sodium hydroxide (1M), and tetrahydrofuran (THF), chromoionophore I, potassium tetrakis [3,5-bis- (trifluoromethyl)phenyl]borate (KTFPB), tetrakis(4- chlorophenyl)borate tetradodecylammonium salt (ETH 500), calcium ionophore IV tris(hydroxymethyl)aminomethane (Tris), acetic acid, sodium acetate were purchased from SigmaAldrich. Dinonylnaphthalene sulfonate (DNNS acid form in 50% heptane) was a gift from King Industry. Aqueous solutions were prepared by dissolving the appropriate salts in Milli-Q-purified distilled water. Protamine stocks solution (10 g L-1) were freshly prepared before starting the experiments. Polypropylene (PP) membranes (Celgard 2500, 25 µm thickness) were obtained from Membrana. FAB (anion-exchanger thickness of 100 µm) membranes were purchased from Fumatech (FuMA-Tech GmbH). PEEK tubing and 1/8 in. and 1/16 in. HPLC connectors were also purchased from Sigma Aldrich. Membrane preparation The cocktail used for both the optimization and rotating disk electrode (RDE) experiments was prepared by dissolving 100 mmol kg−1 of TDDA, 190 mg of o-NPOE and 1 mL of THF. The cocktail for alkalinity and acidity measurements contained 120 mmol kg−1 of chromoionophore I, 60 mmol kg−1 of KTFPB, 90 mmol kg−1 of ETH 500, 190 mg of o-NPOE, and 1 mL of THF. The cocktail for calcium measurements contained 180 mmol kg−1 of calcium ionophore IV, 5 mmol kg−1 of KTFPB, 90 mmol kg−1 of ETH 500 and o-NPOE up to 200 mg of total cocktail amount. For protamine detection, DNNS stock solution was prepared in THF (dry DNNS (112 mg) in THF (1 mL)) and used to prepare the membrane cocktail composed of DNNS (11.8 mg), TDDA (8.82 mg, 2:1 molar ratio respectively), o-NPOE (180 mg), and THF (1 mL). Bare PP membranes were washed with THF for 10 min to remove any possible contaminants. When the membrane was found to be completely dry (in a matter of seconds), an excess volume of 3 µL of the cocktail solution (see above) was deposited on it. The impregnation of the cocktail was found to be instantaneous; however, the membrane was left in the Petri Dish for ca. 10 min to ensure a homogenous and reproducible impregnation of the pores. The pore filling solution composition is assumed to remain identical to the initial THF-free cocktail. Afterwards, the membrane was conditioned in the primary analyte (10 mM) solution for 40 min. The membrane was mounted in the electrode body. All the solutions were prepared in 10 mM of NaCl as background electrolyte, with exception of chloride determination where either none electrolyte or 10 mM NaF was used. The inner compartment containing 5 ACS Paragon Plus Environment


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Ag/AgCl elements was filled with 10 mM solution of each analyte and 10 mM NaCl whereas 100 mM NaCl was used for the RE/CE electrode inner compartment. Flow cell In order to assemble the flow cell, the combined counter/reference electrode (FAB) and working membrane (WE) electrodes were placed into the cell and affixed by screw fittings. The counter/reference (CE/RE) electrode contained a Donnan exclusion membrane (FAB) conditioned in 0.1 M NaCl for 24 h that acted as a separator for a Ag/AgCl element in contact with 0.1 M NaCl. The working electrode was made of porous polypropylene (PP) membrane doped with the membrane cocktail for each analyte (see below for details). The cell was washed with background electrolyte after each measurement (see Figure 1). Electrochemical measurements The electrochemical measurements were performed with a µ-Autolab potentiostat/galvanostat (Metrohm Autolab, Utrecht, The Netherlands) controlled by a personal computer using Nova 1.8 software (supplied by Autolab). The sample was introduced into the flow cell with a peristaltic pump (IPC, ISMATEC) and an automatic injection valve (VICI VALCO, with different loop lengths). The open circuit potential (OCP) was recorded and a 5 to 100 s constant current excitation period was subsequently applied. Before introducing a new sample, the system was regenerated for a period of 45-s at the OCP value previously determined at the beginning of each procedure.12 A sodium chloride solution (10 mM) was used as a carrier to transport the sample from the injection loop to the flow cell. In order to investigate the influence of flow rate on τ, a streaming flow of sample without dilution was introduced to the cell. After optimizing the flow rate, the optimum flow rate was chosen for all subsequent measurement and the plug of the sample was injected into the flow cell by the injection valve.


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RESULT AND DISCUSSIONS Figure 1 schematically illustrates the flow cell design that allows one to perform chronopotentiometry experiments at ion-selective membranes in flow conditions. The flow cell was composed of two electrodes (working and reference/counter) instead of the most common adopted 3-electrode configuration in order to minimize the entire cell and also to reduce the amount of sample (~150 µL). For this reason, counter and reference electrodes were combined and placed opposite the working electrode. The sample solution was pumped through the space between the counter/reference and working electrode (tubular shape with 0.2 mm diameter). The electrodes were located horizontally and the direction of the flow was found to be optimal from the bottom to the top, which avoids air bubbles that would otherwise be easily trapped. A high-density Donnan exclusion ion-exchange material (labelled as FAB) was selected as counter/reference electrode. FAB permits the ion-transport across its pores without appreciable concentration polarization42 and exhibits attractive biocompatibility characteristics.18 For the working electrode, porous polypropylene membrane (PP) doped with plasticizer, ionophore and ion-exchanger was used, as reported earlier.12 The applied current between both electrodes may result in a depletion of the analyte at the surface of the electrode as evidenced by an abrupt change of the potential at a transition time. One would intuitively expect changes in the chronopotentiometric profiles under varying conditions of fluid flow, and an extensive study was here performed to elucidate this behaviour. Preliminary experiments were performed with an anion-exchange membrane (PP membrane doped with TDDA/o-NPOE, 3 mM NaCl and 10 mM NaF as background electrolyte, see experimental section) at different applied currents (from 50 µA to 300 µA) and flow rates (from 0 to 10 mL.min-1). Figure 2a shows the chronopotentiometric data (dE/dt vs. t) in which peaks are easily visualized as result of the local depletion in the entire range of flow rates. It is worth mentioning that there was a slight shift to higher transition times (by less than 0.2 s) at high flow rates for applied currents of 300, 200 and 150 µA. A significant difference in transition times (e.g., from 2s to 5s) was observed at lower flow rates for currents of 100, 75 and 50 µA accompanied with a broadening of the peaks. This is more clearly shown in Figure 2b where the square root of the transition time is plotted as a function of the flow rate for different applied currents. The data suggest that a reduction of the aqueous diffusion layer thickness results in an enhancement of mass transport to the electrode, and therefore, in a time delay of the ion depletion. While this effect was almost negligible for large concentration gradients, it became important for smaller gradients (see Figure 7 ACS Paragon Plus Environment


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S1). To verify these results, they were compared with two different models based on chronopotentiometry at rotating disk electrodes (RDE)13 and in flowing conditions at metal electrodes. 43-44 To mimic the flow chronopotentiometry conditions at the RDE, a PP membrane doped with TDDA/oNPOE (see experimental section) was used as a rotating working electrode in a 100-mL sample, applying the chronopotentiometric protocol at several rotating speeds (from 100 to 150 rpm). Note that ion depletion was not observed at higher rotating speeds for the chosen current amplitudes (Figure S2). The square root of the transition time multiplied by the applied current (τ1/2i) was plotted vs. the imposed current (i) for various rotation speeds (Figure 3a) and fitted with the analytical solution expressed in equation 1 (for details refer to13). ∞   mδ     m 2δ 2  iτ 1/2 nFA π DO  mδ m = × 1+ 2 (−1) exp − − π erfc    ∑ 0     CO 2  DOτ  π DO  π DO    m=1  

−1

(1)

where i is the applied current, τ is the transition time of the chronopotentiogram, C0 is the concentration, n is the charge of the analyte, F is Faraday’s constant, A is the membrane surface area, erfc is the complementary error function, D0 is the ion-diffusion coefficient and δ is the diffusion layer thickness, which can be written in terms of kinematic viscosity (ν), the angular velocity in radians per second (ω) as follows:

δ (cm) =

DO1/3ν 1/6 0.620ω 1/2

(2)

and

ic ≈ 0.606

nFADO π CO0

(3)

δ

The adequate correspondence between experimental data and theory is evidenced in Figure 3. It is anticipated that the hydrodynamic characteristics of rotating ion-exchanger membrane electrodes are analogous to metal electrodes. The observed flow chronopotentiometric data were plotted in the same manner but at varying flow rates (from 0.1 to 1 mL.min-1). As shown in Figure S3, the data exhibit a similar exponential decay as a function of flow rate in comparison to the ones observed in Figure 3.


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Meyer and co-workers

43-44

have developed a model for chronopotentiometric experiments with metal

electrodes in flow conditions. Due to the similarity to the present case, this model was also used to verify the experimental results. Equations 4 and 5 predict the relative transition time (τ/τ0) as a function of ξ, where ξ depends mainly on the flow rate:

 1/2 −1  31/2 ξ   τ 2   1− ξ + 3 tan  = − 2  ln   3ξ   (1+ ξ + ξ 2 )1/2  τ0  2 + ξ 

(4)

ξ = 1.492τ 01/2 D1/2 h −2/3 (UV / dl)1/3

(5)

Here, τ is the transition time of the chronopotentiogram, D is the diffusion coefficient, h is the height of the cell, UV is the streaming flow velocity, d is the width and l the length of the cell. According to this model, increasing the flow rate results in a prolongation of the transition time (τ/τ0), see Figure 4a. Consequently, both the normalized experimental data (markers) and the theoretical predictions (solid line) were plotted vs. the flow rate (Figure 4b). The experimental data are in agreement with theory. Both models (RDE and flow chronopotentiometry) corroborated the observed membrane-based flow chronopotentiometry results and their relationship with the diffusion layer thickness. By changing either the rotation speed or the flow rate, the diffusion layer thickness is varied, resulting in mass transport of variably effectiveness. A compromise between diffusion layer thickness and concentration gradient, as dictated by the applied current, may be found for an intended application. Therefore, this system was optimized for real-world applications in terms of residence time, a range of flow rates from 0.5 to 10 ml min-1 and injection volume. The transition time value (τ) for each injection volume was compared to that for a continuous sample flow without dilution factor. As indicated in Figure 5, different injection volumes were explored, with 150 µL the optimum one for the conditions used in terms of dilution and reproducibility. A 150 µL sample injection volume at 0.5 ml min-1 is a reasonable volume to perform clinical and environmental analysis.4547

This volume is seven times larger than that of the detection cell. The residence time in the detection cell

was calculated as ~15 s by considering the flow rate of 0.5 ml min-1. This is three times longer than the time required (5 s) for performing chronopotentiometry under batch conditions without flow. The analysis of calcium, protamine, chloride, acidity and alkalinity were also performed by flow chronopotentiometry, using dedicated working electrode formulations for each of the analytes (see experimental for details). All measurements were again performed against an anion-exchanger membrane



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(FAB) as counter/reference electrode. The optimum flow rate, residence time, sample volume and pulse duration were 0.5 ml min-1 15 s, 150 µL and 5 s, respectively. Because the determination of free and total calcium concentration has gained attention in biological and clinical studies, calcium under flow conditions was measured using a short current period of 5-s as shown in Figure 6a. The calcium concentration range (from 0.4 mM to 2 mM) and reproducibility (RSD~2%) were found to be adequate for clinical analysis.20 The diffusion coefficient obtained from the slope of the calibration curve (1.21× 10-5 cm2 s-1) confirms the detection of calcium.20 In another example, a symmetrical system based on two Donnan exclusion anion exchange membranes (labelled as “FAB”) was used to measure chloride in flow chronopotentiometry. These hydrophilic materials were previously explored by our group in both potentiometry 42 and batch chronopotentiometry 24

; and recently utilized by Ogawara et al.

48

. Importantly, FAB membranes can be used either as a

universal indicator or working electrode for anions (i.e., nitrate, nitrite, chloride, perchlorate, etc.) since the analytical signal is not strictly dependent of the lipophilicity of the anion of interest.42 Moreover, the observed response agreed with the recent chronopotentiometry theory for Donnan Ion Exclusion Membranes in absence of background electrolyte (pure electrolytes) where migration needs to be considered and therefore results in a increase of the transition time. The results of the flow chronopotentiometric experiments are shown in Figure 6b. The apparent diffusion coefficient (1.52 × 10-4 cm2 s-1) agreed with the reported one under such conditions.24 Under the same flow conditions (0.5 ml min-1), alkalinity and acidity levels were detected by a membrane containing a high concentration of ionophore and ion exchanger.22 Aiming to demonstrate the validity of this approach, the acidity and alkalinity levels were determined in acetic acid (0.2 to 1 mM) and tris base (0.2 to 1 mM), respectively. The time derivative of the chronopotentiograms and calibration curves for both acidity and alkalinity measurements are shown in Figure 7a and b, respectively. The diffusion coefficients for Tris and hydrogen ions (5.82 × 10-6 cm2 s-1; 9 × 10-5 cm2 s-1, respectively) were obtained from the Sand equation as described above. Both values corresponded well with our previous reports under batch conditions.

22-23

It is anticipated that this approach may be useful for the online detection of

carbonate alkalinity in real samples, although thin layer electrochemical approaches are expected to be even more robust.49 The last explored example was the determination of protamine in undiluted human blood. Generally, protamine is post-surgically injected to neutralize heparin concentration administrated during the procedure to control blood clotting time.30-31, 50 This neutralization is quantitative and rapid, allowing one to detect heparin levels by measuring excess protamine.51 10 ACS Paragon Plus Environment

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Our group recently reported on protamine detection by stopped flow chronopotentiometry27 and coulometry19 in blood. The first approach was performed in batch mode using ~25 ml of blood sample, which is unfortunately an excessive volume for clinical diagnostics.

27

For this reason, the propylene

tubular membrane concept, originally reported for calcium by Grygolowicz-Pawlak et al.,52 was adopted for the coulometric detection of protamine and heparin in 5.8 µL of undiluted human blood.19 Unfortunately, the Ag/AgCl element working electrode

19

used in that work is not a biocompatible

material and resulted in an important deterioration of the analytical signal. Building on this experience, a flat sheet membrane

27

instead of tubular one was used here to measure

protamine in the above-mentioned flow cell consuming only 150 µL sample. Figure 8 shows the chronopotentiogram time derivative together with the calibration curve (τ½ vs concentration), which is found to be linear, in agreement with the Sand equation, within the therapeutic concentration range of 20120 mg mL-1.27 The reproducibility of protamine detection was found to be ~2% for 30 measurements by using the same membrane (see Figure S4).



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CONCLUSIONS We demonstrated here the analytical advantages of using flat sheet membranes interrogated by chronopotentiometry in flow conditions for the determination of several ions (i.e., cations, anions and polyions) involved in multiples biomedical and environmental processes. The incorporation of a Donnan ion exclusion membrane as a counter/reference element allowed one to use biological samples without deterioration of the analytical signal due to pollution/adsorption onto the employed materials. The use of 120 µL sample volumes in continuous flow conditions is compatible with clinical diagnostics procedures at a reasonable analytical frequency and guarantees a better reproducibility (typically 1% and 2%) over batch conditions. Even though protamine is reliably measured by using this approach in the range of 20 to 120 ppm, the limit of detection is not yet sufficient for monitoring the back-titration of heparin in human blood. Consequently, efforts to reduce the limit of detection are currently under way in our group. On the other hand, determining alkalinity and acidity by using this approach may be attractive for in-situ determinations in environmental systems. Finally, a direct modulation of the diffusion layer thickness accompanied with a different behaviour in the chronopotentiometry readout is possible by changing the flow rate of the solution. These results were contrasted with two well-established models based on rotating disk electrodes and metal-based flow chronopotentiometry.


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ACKNOWLEDGMENT The authors thank the Swiss National Science Foundation and the European Union (FP7-GA 614002SCHeMA project) for supporting this research. Supporting Information: additional data (chronopotentiograms for variable flow rates at different fixed concentrations; chronopotentiometric time derivatives for a rotating disk electrode as a function of rotation speed and applied current density at fixed sample concentration; transition time analysis as a function of current at varying flow rates; reproducibility of protamine transition time with a single membrane).



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REFERENCES (1)

Le, X. T., Viel, P., Tran, D. P., Grisotto, F., Palacin, S., J. Phys. Chem. B 2009, 113, 5829-5836.

(2)

Rubinstein, I., Maletzki, F., J. Chem. Soc., Faraday Trans 1991, 87, 2079-2087.

(3)

Jaworski, A., Donten, M., Stojek, Z., Anal. Chim. Acta 1995, 305, 106-113.

(4)

Audinos, R., Pichelin, G., Desalination 1988, 68, 251-263.

(5)

Krol, J. J., Wessling, M., Strathmann, H., J. Membr. Sci. 1999, 162, 155-164.

(6)

Choi, J. H., Moon, S. H., J. Membr. Sci. 2001, 191, 225-236.

(7)

Pismenskaia, N., Sistat, P., Huguet, P., Nikonenko, V., Pourcelly, G., J. Membr. Sci. 2004, 228, 65-76.

(8)

Lee, H. J., Hong, M. K., Han, S. D., Moon, S. H., J. Membr. Sci. 2008, 320, 549-555.

(9)

Bodor, S., Zook, J. M., Lindner, E., Toth, K., Gyurcsanyi, R. E., J. Solid State Electrochem. 2009, 13, 171-179.

(10)

Zook, J. M., Bodor, S., Lindner, E., Toth, K., Gyurcsanyi, R. E., Electroanalysis 2009, 21, 19231930.

(11)

Klaysom, C., Moon, S. H., Ladewig, B. P., Lu, G. Q. M., Wang, L. Z., J. Membr. Sci. 2011, 371, 37-44.

(12)

Crespo, G. A., Bakker, E., Rsc Advances 2013, 3, 25461-25474.

(13)

Buck, R., Keller, H., Anal. Chem. 1963, 35, 400-402.

(14)

Bard, A. J., Anal. Chem. 1963, 35, 1602-1607.

(15)

Bard, A. J., Anal. Chem. 1961, 33, 11-&.

(16)

Bard, A. J., Faulkner, L. R., Electrochemical Methods: Fundamentals and Applications. New York, 2001.

(17)

Abbas, Y., Olthuis, W., van den Berg, A., Sens. Actuators B Chem. 2013, 188, 433-439.

(18)

Afshar, M. G., Crespo, G. A., Bakker, E., Biosens. Bioelectron. 2014, 61, 64-69.

(19)

Crespo, G. A., Afshar, M. G., Dorokhin, D., Bakker, E., Anal. Chem. 2014, 86, 1357-1360.


Page 15 of 26

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(20)

Ghahraman Afshar, M., Crespo, G. A., Bakker, E., Anal. Chem. 2012, 84, 8813-8821.

(21)

Jansod, S., Afshar, M. G., Crespo, G. A., Bakker, E., Biosens. Bioelectron. 2016, 79, 114-120.

(22)

Crespo, G. A., Ghahraman Afshar, M., Bakker, E., Anal. Chem. 2012, 84, 10165-10169.

(23)

Afshar, M. G., Crespo, G. A., Xie, X., Bakker, E., Anal. Chem. 2014, 86, 6461-6470.

(24)

Crespo, G. A., Ghahraman Afshar, M., Bakker, E., J. Electroanal. Chem. 2014, 731, 100-106.

(25)

Jarolimova, Z., Crespo, G. A., Afshar, M. G., Pawlak, M., Bakker, E., J. Electroanal. Chem. 2013, 709, 118-125.

(26)

Jarolimova, Z., Crespo, G. A., Xie, X., Afshar, M. G., Pawlak, M., Bakker, E., Anal. Chem. 2014, 86, 6307-6314.

(27)

Crespo, G. A., Ghahraman Afshar, M., Bakker, E., Angew. Chem. Int. Ed. Engl. 2012, 124, 12743-12746.

(28)

Bakker, E., Crespo, G. A., Afshar, M. G., Saxer, T., Bendjelid, K., Chimia 2013, 67, 350-350.

(29)

Frank, R. D., Brandenburg, V. M., Lanzmich, R., Floege, J., Nephrol. Dial. Transplant. 2004, 19, 1552-1558.

(30)

Khaja, W. A., Bilen, O., Lukner, R. B., Edwards, R., Teruya, J., Am. J. Clin. Pathol. 2010, 134, 950-954.

(31)

Vandiver, J. W., Vondracek, T. G., Pharmacotherapy 2012, 32, 546-558.

(32)

Ma, S. C., Yang, V. C., Meyerhoff, M. E., Anal. Chem. 1992, 64, 694-697.

(33)

Ma, S. C., Yang, V. C., Fu, B., Meyerhoff, M. E., Anal. Chem. 1993, 65, 2078-2084.

(34)

Fu, B., Bakker, E., Yun, J. H., Yang, V. C., Meyerhoff, M. E., Anal. Chem. 1994, 66, 2250-2259.

(35)

Yun, J. H., Meyerhoff, M. E., Yang, V. C., Anal. Biochem. 1995, 224, 212-220.

(36)

Ramamurthy, N., Baliga, N., Wahr, J. A., Schaller, U., Yang, V. C., Meyerhoff, M. E., Clin. Chem. 1998, 44, 606-613.

(37)

Ramamurthy, N., Baliga, N., Wakefield, T. W., Andrews, P. C., Yang, V. C., Meyerhoff, M. E., Anal. Biochem. 1999, 266, 116-124.

(38)

Chen, Y., Ding, J., Qin, W., Analyst 2012, 137, 1944-1949. 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

(39)

Lei, J., Ding, J., Chen, Y., Qin, W., Anal. Chim. Acta 2015, 858, 60-65.

(40)

Bell-Vlasoy, A. K., Zajda, J., Eldourghamy, A., Malinowska, E., Meyerhoff, M. E., Anal. Chem. 2014, 86, 4041-4046.

(41)

Gemene, K. L., Meyerhoff, M. E., Anal. Chem. 2010, 82, 1612-1615.

(42)

Grygolowicz-Pawlak, E., Crespo, G. A., Ghahraman Afshar, M., Mistlberger, G., Bakker, E., Anal. Chem. 2013, 85, 6208-6212.

(43)

Meyer, R., Banta, M., Lantz, P., Posey, F., J. Electroanal. Chem. Interfacial. Electrochem. 1971, 30, 345-358.

(44)

Posey, F., Meyer, R., J. Electroanal. Chem. Interfacial. Electrochem. 1971, 30, 359-373.

(45)

Masoom, M., Townshend, A., Anal. Chim. Acta 1985, 166, 111-118.

(46)

Ruzicka, J., Hansen, E. H., Flow injection analysis. John Wiley & Sons: 1988; Vol. 62.

(47)

Hansen, E., Ghose, A. K., Růžička, J., Analyst 1977, 102, 705-713.

(48)

Ogawara, S., Carey, J. L., Zou, X. U., Bühlmann, P., ACS Sensors 2015,

(49)

Afshar, M. G., Crespo, G. A., Bakker, E., Angew. Chem. Int. Ed. Engl. 2015,

(50)

Raymond, P. D., Ray, M. J., Callen, S. N., Marsh, N. A., Perfusion-Uk 2003, 18, 269-276.

(51)

Whitelock, J. M., Iozzo, R. V., Chem. Rev. 2005, 105, 2745-2764.

(52)

Grygolowicz-Pawlak, E., Bakker, E., Anal. Chem. 2010, 82, 4537-4542.


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FIGURE CAPTIONS Figure 1. Schematic illustration of the custom-made flow chronopotentiometric cell. The working electrode (WE) is located opposite the counter/reference electrode (CE/RE). After the sample is injected, the chronopotentiometric protocol is started once the sample plug arrives in the cell, by applying the current between the two electrodes without stopping sample flow. Figure 2. a) Effect of the flow rate on the observed chronopotentiograms within the range of 0.5 to 10 mL.min-1). b) Flow rate as a function of transition time (τ) for different current amplitudes (300, 200, 150, 100, 75 and 50 µA) under flow conditions. A PP membrane doped with 100 mM TDDA and plasticizer was used as working electrode. The sample contained 3 mM NaCl in 10 mM NaF as background electrolyte. Figure 3. Plot of iτ1/2 as a function of the applied current (i) at several rotating speeds within the range of 100 to 150 rpm. Each dataset (marker) was fitted with equation 1 (solid line). A PP membrane contained 100 mM TDDA was used as working membrane. The sample contained 3 mM NaCl in 10 mM NaF as a background electrolyte. Fitting parameters for eq 1 were: n=1, F=96485, A=0.21 dm2, C=3 mM, i=300 µA, and D=1.5×10-6 cm2s-1.13 Figure 4. a) Relative transition time as a function of parameter ξ. Parameters: h=1.5 mm, dl=9.30 mm2 corresponds to the circular area of the electrode, C=3 mM, i=300 µA, z=1 and D=1.5×10-6 cm2s-1. b) Effect of flow rate on relative transition time for various current amplitudes. Each dataset (marker) was fitted with equation 4 (solid line).43-44 Figure 5. Effect of the injected volume on the chronopotentiometric response of a protamine-selective membrane electrode (100 ppm protamine in 100 mM NaCl, applied current of 2 µA for 5-s). The red line shows the transition time for a flowing sample solution without dilution effect. Black solid lines are for different injection loop volumes. Figure 6. a) Chronopotentiometric time derivatives for a calcium-selective membrane when successively increasing the final calcium concentration from 0.4 to 2 mM. Time derivatives for chloride detection within the range of 0.2 to 0.8 mM. Inset: Linear calibration curve of the square root of the transition time (τ1/2) as a function of concentration. The current pulses for calcium and chloride detection were -50 and 350 µA, respectively. Figure 7. a) Chronopotentiometric time derivatives for total acidity measurement with a sample containing 0.2−1 mM of acetic acid. b) Time derivatives for alkalinity measurement in contact with a sample containing 0.2−1 mM of tris base. Inset: Linear calibration curve of the square root of the transition time (τ1/2) as a function of acidity and alkalinity, respectively. The current pulses for acidity and alkalinity detection were -14 and 12 µA, respectively. Figure 8. a) Chronopotentiometric time derivatives for protamine selective membranes in undiluted human blood with increasing protamine concentration from 20 to 100 mg L-1.



Fig. 1 OUTLET

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CE/RE

WE

INLET

ACS Paragon Plus Environment

Page 18 of 26

Page 19 of Fig. 26 2


10

FLOWRATE / mL min -1

a) TIME DERIVATIVE OF CHRONOPOTENTIOGRAM 300 µA

8

INCREASING FLOW

[dE/dt] / mV s -1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6

200 µA INCREASING FLOW

150 µA

4

INCREASING FLOW

2

0 0

1

2

3

4

5

t/s

12 b) FLOWRATE VS t

10

300 µA 200 µA 150 µA 100 µA 75 µA 50 µA

8 6 4 2 0 0

2

4

6 1/2

t /s

8

10

1/2



Fig. 3

1/2

It /m As

-1/2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

380

ROTATING DISK ELECTRODE

360

RPM

150 140 130 140 110 105 100

340 320 300 280 260 240 150

200

250

300

350

I/m A


Page 20 of 26



1.2

a) RELATIVE t VS ?

1.15

t /t

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0

1.1

1.05

1 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

?

b) FLOW CHRONOPOTENTIOMETRY 2.2

300 µA 200 µA 150 µA 100 µA 75 µA 35 µA

1.8

0

t /t

2

1.6 1.4 1.2 1 0.8

0

2

4

6

FLOWRATE / mL min

8

10

-1



Fig. 5 1

[dE/dt] / mV s -1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OPTIMIZATION OF THE INJECTED VOLUME 50 µl 60 70

0.8

80 90

0.6

100 120 130140

0.4 150

0.2

0

0

1

2

3

4

5

t/s


Page 22 of 26



5

[dE/dt] / mV s -1

a) CALCIUM

2.5 2

1/2

4 1/2

t /s

0.4 mM

1.5 1 0.5

3

0

0.5

0

1

1.5

2

2.5

2+

[Ca ] / mM

0.8 mM 2

1.2 mM 1.6 mM

1

2 mM

0

0

1

2

3

4

0.2

0.4 0.6 [Cl ] / mM

5

t/s 35

b) CHLORIDE 2

0.2 mM

30

1/2 1/2 t /s

[dE/dt] / mV s

-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.3 mM

25

1.6 1.2 0.8

20

0.4

0.4 mM 15

0.8

0.5 mM 0.6 mM

10

0.7 mM

0.8 mM

5

0

0

1

2

3

4

5

t/s



Fig. 7 7

a) ACIDITY 6

2 1/2 1/2 t /s

0.2 mM

[dE/dt] / mV s

-1

5

0.4 mM

4

1.5 1 0.5 0.4

0.2

3

0.6 0.8 ACIDITY / mM

1

0.6 mM 0.8 mM

2

1 mM

1

0

0

1

2

3

4

5

t/s 6

a) ALKALINITY 2

5 t /s

1/2

1.5

1/2

0.2 mM

4

[dE/dt] / mV s -1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 0.5 0

3

0.2

0.4 mM 2

0.4

0.6

0.8

1

ALKALINITY / mM

0.6 mM

0.8 mM 1 mM

1

0

0

1

2

3

4

5

t/s


Page 24 of 26

Page 25 of Fig. 26 8 PROTAMINE DETECTION IN WHOLE BLOOD 2

20 ppm 1.5 1/2 1/2 t /s

1.5

40 ppm

[dE/dt] / mV s

-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 ppm

Y=0.0108X+0.3531

1

0.5 0

20

40 80 100 60 [PROTAMINE] / ppm

120

80 ppm 0.5

100 ppm 120 ppm

t/s



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC Graphic 92x133mm (300 x 300 DPI)


Page 26 of 26

Flow Chronopotentiometry with Ion-Selective Membranes for Cation

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