Different morphologies of polypyrrole produced by flow-through and batch electropolymerizations: application in electrochemically controlled in-tube solid phase microextraction

Abstract

In this paper, polypyrrole (PPy) was electropolymerized with perchlorate ion on the inner surface of stainless steel tubes (in-tube) and also on stainless steel wires. A batch electropolymerization and three modes of in-tube flow-through electropolymerization were investigated. Electrosynthesis of PPy-ClO₄ was observed in all the mentioned modes, ascertained from the similarity of the infrared spectra of their products, despite their quite different morphologies. According to the position of the counter electrode (relative to working electrode) and the geometry of the working electrode, different morphologies and thicknesses can be produced. Furthermore, the PPy tube formed by the flow-through electropolymerization was used in electrochemically controlled in-tube solid phase microextraction (EC in-tube SPME). EC in-tube SPME decreased the total analysis time and increased the sensitivity. It was found that the extraction efficiency could be greatly enhanced by using a constant potential.

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Introduction

Conducting polymers, particularly PPy and its derivatives, have attracted a great deal of attention due to their multifunctional properties (e.g., hydrophobicity, acid–base character, π–π interaction, polar functional groups, ion-exchange property, hydrogen bonding, and electroactivity).¹ PPy has been used in many different areas such as ion-exchange, energy-storage materials, corrosion-resistant coatings, catalysts, and materials for separation, chemical sensors and biosensors, and the so-called “electronic nose”.² Pyrrole can be polymerized using oxidation reactions by either an electrochemical or a chemical method.³ In several studies, chemical polymerization of pyrrole on the inner surface of a capillary (in-tube) has been reported.^1,4 Briefly, the PPy inner surface coating was prepared by first passing the monomer solution and then the oxidant solution through the capillary with the aid of nitrogen gas. The above procedure was referred to as one PPy coating cycle, which could be repeated several times to increase the thickness of PPy coating. Limitations encountered with this method include: (1) due to less interactions between PPy and capillary, adhesion of PPy to capillary is not strong and this affect the mechanical stability of polymer, (2) the electrical conductivity of PPy formed by chemical method is often low and although development of electrical conductive PPy by chemical polymerization are reported,⁵ but these PPy were not used in SPME (or in-tube SPME), (3) the number of dopants in chemical polymerization method are very limited, (4) physical properties of the synthesized polymer cannot easily be controlled. Due to the electrodepositing of PPy on the surface of metals from an aqueous solution containing pyrrole and electrolyte, electrochemical synthesis is more convenient. The physical properties of the polymer can be controlled more easily with electrochemical methods. In addition, using different substituted pyrrole monomers or various kinds of dopant ions, along with controlled electrochemical conditions, could result in the formation of PPy with flexible characterizations. For electrochemical synthesis, conducting electrodes such as stainless steel are required. The PPy film was directly prepared on the surface of the working electrode from a electrolyte solution containing defined pyrrole monomer and dopant by applying a constant deposition potential or a constant deposition current.⁶ The experimental variables influenced electrochemical polymerizations are monomer, solvent, counter ion, and electrode materials. These variables affect the physical properties of PPy such as morphology which is important factor that influence uptake behavior of PPy sorbent. In previous report, we electrosynthesized molecularly imprinted PPy film on the surface of stainless steel wire as a selective sorbent for benzoate anion. We demonstrated that dopant (counter ion) has important role in morphology of PPy film.⁷ To the best of our knowledge, the effect of working electrode geometry (wire or tube) and counter electrode position in the morphology of PPy film has not been investigated so far. In current work, PPy film was elctropolymerized with perchlorate ion in the tube and also on the wire. We investigated three new modes of in-tube electropolymerization and a mode of batch electropolymerization.

For the analysis of analytes in complex matrices, sample treatments such as extraction, preconcentration and clean-up steps are often required to improve the sensitivity and selectivity. Electrochemically controlled solid-phase microextraction (EC-SPME) combines SPME with electrochemistry.^7,8 EC-SPME use conducting polymers, whose charge can be electrochemically controlled by oxidation or reduction, as the stationary phase. In current study, the electropolymerized tube was used in the in-tube SPME. This tube acted as working electrode for absorption of diclofenac as target analyte. Extraction ability of the PPy-coated tube with and without applied electrical field was investigated.

Experimental section

Chemicals and materials

Sodium diclofenac and other materials were purchased from Merck (Darmstadt, Germany). All solvents were HPLC grade and were purchased from Sigma-Aldrich (Steinheium, Germany). Milli-Q water (Millipore, Billerica, MA, USA) was used to prepare the samples. Proper amount of sodium diclofenac was dissolved in methanol to obtain stock solution of the analyte with a concentration of 1 mg mL⁻¹. Working standard solutions were freshly prepared by diluting the standard solution of the analyte with ultra-pure water to the required concentration. Pyrrole was obtained from Merck (Darmstadt, Germany), distilled and kept under nitrogen in darkness at 4 °C before use. pH was adjusted with 0.1 M sodium hydroxide and hydrochloric acid solutions.

Electrosynthesis of PPy on the inner surface of stainless steel tube

Before synthesis, the stainless steel tubes (10 cm × 0.8 mm i.d.) were cleaned with a mixture of acetone and pure water (70 : 30, v/v). This solution was percolated through the tubes for 10 min, and then the tubes thoroughly rinsed with water, acetone and water, respectively. A solution of 0.1 mol L⁻¹ pyrrole and 0.2 mol L⁻¹ LiClO₄ was filled into container, purged with N₂ gas for 2 min, and then used as mother solution for electrosynthesis of PPy in tube. Three modes of flow electropolymerization were investigated. In the first mode, the tube (working electrode) and the zero-dead-volume internal union (counter electrode) were consecutively placed (method 1). Insulating materials were placed between these two electrodes (Fig. 1a). In the second and third modes, a very thin copper wire (15 cm × 0.07 mm diameter) was placed into the tube (Fig. 1b). The tube and the wire serve as working and counter electrodes, respectively. Insulating material was used to prevent electrical connection between these electrodes. In method 2, the peristaltic pump (Master Flex, model 7013-20, Chicago, IL, USA) was turned on during the whole electropolymerization time whereas in method 3, the pump was turned on and off for 30 and 20 s, respectively and this program was repeated for the whole electropolymerization time (300 s). In all modes, by passing the solution through the tube and applying a constant current of 3 mA, electrochemical polymerization of pyrrole was carried out. For PPy-coated tube formed by method 1, the stainless steel tube was washed with methanol to remove the unreacted component. However, for PPy-coated tube formed by methods 2 and 3, the counter electrode was removed and then the stainless steel tube was washed with methanol. Finally, PPy-coated tube produced by method 3 was fixed in the place of the injection loop of the HPLC system. When the tube was not in use, it was filled by 0.01 mol L⁻¹ LiClO₄ at room temperature.

Fig. 1 Schematic Illustration of setup for electropolymerization of PPy: (a) method 1; (b) methods 2, 3; (c) batch electropolymerization. (1) Power supply, (2) polymerization solution, (3) flow pump, (4) waste solution, (5) insulating materials. WE and CE indicate working and counter electrodes, respectively.

Electrosynthesis of PPy on the stainless steel wire

Before synthesis, the surface of a stainless steel wire (2 cm × 1 mm diameter) was washed with a mixture of acetone and pure water (70 : 30, v/v) for 10 minutes in ultrasonic bath. PPy film was coated on the stainless steel wire (working electrode) by an electrochemical polymerization method described previously with some modifications.⁷ Briefly, polymerization of pyrrole was carried out in a nitrogen-purged aqueous solution containing 0.1 mol L⁻¹ pyrrole and 0.2 mol L⁻¹ perchlorate by applying a constant current of 3 mA (Fig. 1c). When the PPy fiber was not in use, it was stored in 0.01 mol L⁻¹ LiClO₄ at room temperature.

Scanning electron microscopy (SEM)

The PPy-coated tubes and wire were cut into a 1 cm long pieces and then analyzed using a VEGA3 TESCAN scanning electron microscope (Brno, Czech) with 20 kV accelerating potential.

Instrumentation and analytical conditions

Separation and detection of the diclofenac was performed by a Knauer HPLC containing a K-1001 well chrome HPLC pump, a six-port HPLC valve with a 20 μL sample loop and a K-2600 UV-vis detector. Chromatographic data were recorded and analyzed using Chrom Gate software (Knauer), version 3.1. The separation was carried out on a C18 column (250 mm × 4.0 mm, with 5 μm particle size). The mobile phase consisted of 10 mM phosphate buffer, pH 3, and methanol (80 : 20). The flow rate of mobile phase was set at 1.0 mL min⁻¹. Detection was performed at the wavelength of 280 nm. Electrochemical experiments were performed using a Behpajuh (BHP 2064+ model) potentiostat (Isfahan, Iran). All experiments were carried out at ambient temperature. All pH measurements were performed at 25 ± 0.1 °C using a pH-meter Metrohm 780 with a standard uncertainty of 0.1 mV (Metrohm, Switzerland).

EC in-tube SPME-HPLC

The stainless steel tube was fixed in the place of the injection loop of the HPLC system. To complete the three-electrode setup, two zero-dead-volume internal unions were used as counter and reference electrodes (Fig. 2). Insulating materials were used to prevent short-circuit connection between these three electrodes. The in-tube SPME-HPLC consisted of two segments: chromatographic (HPLC) and in-tube SPME segments which a six-port injection valve was used to join them. The main parts of in-tube SPME segments included of a three-electrode setup, an electrochemical device and a flow pump. 1/16 in. stainless steel and polyether ether ketone (PEEK) nuts and ferrules were used to complete the connections. Before use, the PPy-coated tube was washed and conditioned by ultrapure water. As shown in Fig. 2 (LOAD), the electroextraction was performed by passing the sample solution (15 mL) through the stainless steel tube using the flow pump. At this time, a positive three-electrode constant potential was applied. In load position, the mobile phase was driven by HPLC pump through the analytical column to obtain a flat baseline in preparation for chromatographic separation. To prevent contamination of the analytical column with residual compounds, the PPy-coated tube was washed with water (1 mL). Then, 1 mL of 10 mM buffer solution, pH of 3, and acetonitrile (50 : 50, v/v) was percolated into the tube and desorption of the analyte was carried out in static mode with application of negative potential. After that, the valve switched to inject position (Fig. 2 (INJECT)) and the desorbed analyte was introduced to HPLC.

Fig. 2 Schematic of the EC in-tube SPME-HPLC setup.

Sample preparation

Urine samples were collected from drug-free, healthy volunteer. Any precipitated material was removed by centrifuging the sample at 4000 rpm (Hettich, Rotofix 32A) for 10 min. The supernatant of urine samples were directly spiked with diclofenac, diluted 5 times with water, and adjust to pH of 9 with NaOH.

Results and discussions

Electrosynthesis of PPy

To investigate the effect of counter electrode position on the thickness of polymeric film of PPy-coated wire and tube, three new modes of flow-through and a batch electropolymerizations were investigated. In all modes, electropolymerization time was kept constant at 300 s. Some laboratory setups employ a two-electrode batch setup to maximize the reproducibility of the polymerization process. The positioning of the auxiliary electrode is critical in that it determines the electrical field generated, which can influence the quality and evenness of the polymer deposited. In our study, with increasing the distance between the working and the counter electrodes greater than 5 cm, the thickness of PPy changed dramatically (Table 1). The adhesion of PPy to the electrode surface is another consideration, hence, the positioning of the auxiliary electrode is of paramount importance. In method 1, in which the counter and the working electrodes were consecutively placed, three distances were investigated. With increasing the distance between the working and the counter electrodes up to 5 cm, the thickness of PPy did not change significantly. The vicinity of the two electrodes and the movement of the solution cause fast moving the electrical charge in the solution and forming PPy in the tube. In distances greater than 5 cm, the thickness gradually reduced so that at distance of 10 cm, the sorbent did not formed. Furthermore, by keeping the distance at 1 cm and by changing the length of the tube (the working electrode), it was observed that the PPy thickness in the tube was the same up to 5 cm and at greater distance the thickness was gradually decreased. Therefore, method 1 is appropriate for producing PPy-tube with length up to 5 cm. To produce PPy-tube with length greater than 5 cm, a thin wire (counter electrode) was placed inside the tube (the working electrode). In method 2, the electropolymerization was performed in several lengths of the tube but, as can be seen in Table 1, the thickness of PPy was low. Wallace et al. were reported the producing of colloids or water-soluble PPy using a developed flow-through electrochemical cell.⁹ Furthermore, Li et al. used the hydrodynamic system to produce non-deposited PPy which was formed at the anode and continuously removed from the cell in the form of fibers.¹⁰ In current work, we also have used stopped flow steps (method 3) to form deposited PPy in the tube. In this method, the pump was kept on for 30 s and after that it was switched off for 20 s. The stopped flow process was repeated for the whole electropolymerization time (300 s). When the pump was switched off, due to the vicinity of two electrodes and the movement of electrical charge (ions), pyrrole monomers deposited in the tube as PPy. The cell hydrodynamics regulate the movement of reactants and products to/from the electrode. With respect to the rate-determining step, the electrochemical reactions occurring at the cathode cannot be ignored. Particularly in a two-electrode cell, this electrochemical step may become the rate-determining factor. This is the case, for example, when reduction of water is the cathodic reaction. Therefore, the hydrodynamics of the electrochemical system are important because these control the rate of transport of reactants and products to and from the electrochemical reaction zone. This in turn determines the polymerization efficiency. When the pump was switched on, gas formed in the counter electrode and other impurities left the tube. In method 3, regardless of the length of the tubes, PPy polymers with the same thickness can be produced (Table 1).

Table 1 Effect of thickness variations with respect to the position of counter electrode and the length of the tube^a

Mode	Distance/thickness	Length/thickness^b
a Both the distance and the thickness are in cm and the length is in μm.b Thickness at the end of the tube.
Batch	1/23	—
	5/19	—
	10/8	—
Method 1	1/12	1/12
	5/10	5/11
	10/—	10/—
Method 2	—	1/5
	—	5/5
	—	10/4
Method 3	—	1/18
	—	5/18
	—	10/18

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The nature and concentration of the dopant, electrode substrate, solvent, polymerization time and current influence the morphology of PPy films.¹¹ Porosity of the film surface and the size of the inclusion sites vary under different polymerization conditions. The morphologies of the PPy-coated tube (method 3) and wire which investigated by scanning electron microscopy (SEM) are interesting. The most common morphology of PPy is the cauliflower structure,¹² but the surface roughness and porosity depend on the experimental conditions used in fabricating the polymer and these can lead to changes in the cauliflower morphology. As can be seen from Fig. 3(a) and (b) the morphology of PPy-ClO₄ coated on the stainless steel wire is cauliflower, whereas the morphology of PPy-ClO₄ coated on inner surface of stainless steel tube is filamentous. In batch electropolymerization, at first PPy formed on the opposite side (relative to the counter electrode) of the wire and after that there would be two possibilities: (1) growing this PPy towards the counter electrode, and only the opposite side of the wire will be polymerized and the probable morphology of this PPy will be filamentous; (2) continuing the growth of this PPy in all sides of the tube. In the second possibility, the probable morphology of PPy will be uniform and homogeneous. Due to the distance between two electrodes and the tendency of PPy to grow more, the observed morphology for the PPy-coated wire could be explained on the basis of theory No. 2. As can be seen from Fig. 3(b), the morphology of PPy-ClO₄ coated on the stainless steel wire is uniform, homogeneous and cauliflower. In flow through electropolymerization (method 3), because all sections inside the tube had equal distance from the counter electrode (the copper wire inside the tube) and also the distance between two electrodes was small, pyrrole monomers selected path no. 1 to carry out the polymerization. PPy grew toward the counter electrode which placed inside the stainless steel tube and the morphology of PPy-ClO₄ coated on inner surface of stainless steel tube was filamentous (Fig. 3(a)). Therefore, according to the place of counter electrode (relative to working electrode) and the geometry of working electrode (tube or wire), different morphologies can be produced. SEM images also show that the PPy were homogeneously distributed in the stainless steel tube and on the wire (Fig. 3(c) and (d)). The morphology and quality of a polymeric film is one of the critical features for its ability to serve as a SPME device.

Fig. 3 The SEM of PPy-coated tube (a) and (c) and wire (b) and (d) with magnification of (a) 4.17k×, (b) 844×, (c) 173×, and (d) 187×.

FT-IR spectra of PPy-coated on the wire and in-tube (method 3) are shown in Fig. 4. N–H, C=C, and C=N stretching bands for PPy are the same for on the wire and in the tube as expected.

Fig. 4 FT-IR spectra of PPy electrosynthesize on the wire and in the tube (stretching bands).

Configuration of EC in-tube SPME-HPLC

Before performing EC in-tube SPME and to insure that this setup worked properly, two experiments were designed. A 0.01 mol L⁻¹ lithium perchlorate solution was passed through the system and cyclic voltammetry (CV) with scan rate of 50 mV s⁻¹ was applied. Furthermore and for the comparison, a PPy stainless steel fiber was immersed in a batch three-electrode cell and the same CV technique was applied. In batch experiment, the counter and the reference electrodes were the same with flow-through experiment. The reversibility of the reaction is important to quantitatively recover the extracted analyte and ensuring no sample carry-over from previous extractions. As can be seen in Fig. 5, the cyclic voltammogram obtained by the two experiments were approximately the same. Two important notes can be extracted from the results: (1) the PPy-coated tube was properly uptake and release perchlorate anion, and (2) there are electrical connections between three electrodes. After ensuring the electrical connections, a 100 ng mL⁻¹ solution of diclofenac was passed through the system. The conductivity of the solution was low, therefore for increasing the conductivity, LiClO₄ salt was added.

Fig. 5 Cyclic voltammograms of 0.01 mol L⁻¹ LiClO₄ solution at scan rate 50 mV s⁻¹ obtained by flow-through (dash-line) and batch (line) experiments.

Uptake and release potentials. In all experiments flow rate, uptake volume, uptake time, release time, pH of uptake solution were kept constant at 0.9 mL min⁻¹, 15 mL, 10 min, 6 min and 9, respectively. Accumulation and release were achieved by applied positive and negative potentials, respectively. During the oxidation of polypyrrole, the positive charges were formed on the PPy chain and were used to extract the anions from the solution. In the next step, the charge of the PPy was neutralized and therefore the anions were released. The influence of the potential used during the diclofenac uptake and release steps is shown in Fig. 6(a) and (b). The potentials of +1.2 and −0.4 V versus stainless steel electrode as pseudo-reference for uptake and release, respectively, were found to give the highest responses.

Fig. 6 Effect of the uptake (a) and release (b) potentials on the extraction of diclofenac. (c) The anion uptake and release properties of the PPy-coated tube under open circuit (in-tube SPME) and controlled potential (EC in-tube SPME) conditions. The concentration of diclofenac solution was 0.1 μg mL⁻¹.

Furthermore, the anion uptake and release properties of the PPy-coated tube was investigated for diclofenac under both open circuit (in-tube SPME) and controlled potential (EC in-tube SPME) conditions (Fig. 6c). Three separate experiments were designed for this purpose employing in-tube SPME: (1) the diclofenac was absorbed in the PPy and the concentration gradient of diclofenac between solution and PPy was the driving force. In this case, the absorption efficiency was very low; (2) a potential was applied only in the uptake process, then both applied potential (in uptake process) and concentration gradient (in release process) acted as driving force; (3) a potential was applied only in release process and the amount of diclofenac absorbed to the tube was decreased. Despite of applied potential in release process, only small amount of diclofenac was released. As a result, applied potential in uptake process was more effective than that of release process.

Precision, limit of detection, and linearity

The repeatability (run-to-run RSD) and the reproducibility (tube-to-tube RSD) of the EC in-tube SPME-HPLC were calculated over eight analyses of diclofenac with PPy-coated tube (Table 2). The concentration level for calculation of the RSD is 50 μg L⁻¹. The PPy-coated tube has a good mechanical stability and can be used for 50 times without a tangible change in stability and extraction efficiency. After 50 times use of this tube, the RSD was increased to 7.3% for 8 replicate analyses of 50 μg L⁻¹ solution of diclofenac. The EC in-tube SPME method can enhance sensitivity more than 14 and 20 times relative to in-tube SPME and direct injection method (25 μL injection), respectively. The LOD was calculated according to the formula 3σ_b/m, where m is the slope of the calibration curve and σ_b is standard deviation of the blank signal.

Table 2 Figures of merit of the proposed EC in-tube SPME-HPLC method^a

Regression equation	Linearity		LOD^c	Precision (RSD, n = 8)
	LDR^b	R^2		Run-to-run	Tube-to-tube
a All concentrations are in μg L^−1.b Linear dynamic range.c Limit of detection.
y = 602.26x − 1596	0.5–1000	0.9995	0.1	4.7	5.9

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Analysis of urine samples

The applicability of PPy-coated tube for EC in-tube SPME-HPLC was investigated for urine samples. The recoveries of the diclofenac was 94.4–97.2% (Table 3). As shown in Fig. 7a, no interference peaks were observed in non-spiked urine sample (sample 1). Fig. 7b shows typical chromatogram, obtained after EC in-tube SPME-HPLC of spiked urine sample.

Table 3 Performance of the proposed method for extraction of naproxen from urine samples

Sample	Added (μg L^−1)	Found (μg L^−1)	RSD (n = 8)	Recovery (%)
Urine (1)	25	23.7	7.12	94.8
	100	94.9	5.23	94.9
Urine (2)	25	24.3	4.23	97.2
	100	96.4	6.76	96.4
Urine (3)	25	23.9	7.90	95.6
	100	94.4	5.21	94.4

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Fig. 7 HPLC chromatograms of diclofenac in urine before spiking (a) and after spiking with 0.1 μg mL⁻¹ (b) using EC in-tube SPME method combined with HPLC-UV under optimum conditions.

Conclusion

In previous report, the significance of dopant in morphology and uptake ability of PPy-sorbent were demonstrated.⁷ In current work, PPy film was electropolymerized with perchlorate ion on the inner surface of stainless steel tube (in-tube) and also on the stainless steel wire. Electrosynthesis of PPy-ClO₄ was observed in the all mentioned modes from the similarity of infrared spectra of their products, despite their quite different morphologies. The morphology of PPy-ClO₄ coated on the stainless steel wire is cauliflower, whereas the morphology of PPy-ClO₄ coated on inner surface of stainless steel tube (method 3) is filamentous. We demonstrated that the film morphology was strongly dependent on the geometry of working electrode and the position of the counter electrode. The morphology and quality of a polymeric film is one of the determining features for its ability to serve as a SPME device. If length of the tube is less than 5 cm, and because of simplicity in operation, method 1 is more preferred. In lengths greater than 5 cm, method 3 produce homogeneous PPy film and therefore this method is more convenient.

The PPy-coated tube was used in the EC in-tube SPME-HPLC for the determination of diclofenac in urine samples. It has been demonstrated that the application of a constant electric potential to the PPy-coated tube significantly improves the extraction efficiencies of diclofenac. This method can be extended to the analysis of other groups of analytes with little modification.

Acknowledgements

The Research Council of Chemistry and Chemical Engineering Research Center of Iran (CCERCI) is acknowledged for supporting the project. The authors express their sincere appreciation to Dr Kourosh Tabar Heydar for scientific advices.

Notes and references

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