Constant current chronopotentiometric stripping analysis of “N-catalyst” in sodium chloride solution and seawater
Slađana Strmečki*, Marta Plavšić, Božena Ćosović
Ruđer Bošković Institute, Division for Marine and Environmental Research,
Bijenička 54, P.O.Box 180, 10 002 Zagreb, Croatia
* Corresponding author, e-mail:
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Abstract
Catalytic properties and surface activity of nitrogen containing polymeric organic material (N-POM) were analyzed by constant current chronopotentiometric stripping analysis (CPSA) and alternating current (AC) voltammetry in sodium chloride solution (pH 8) and seawater. CPSA proved to be a suitable method for determination of low concentrations of N-POM in seawater by measuring its “presodium” catalytic peak H. A protein human serum albumin (HSA) (15 % of N) was used as a model compound and the concentration of N-POM from natural seawater samples was expressed in HSA concentration equivalents. Peak H represents an additional parameter for characterization of natural organic matter.
Keywords: N-containing polymeric organic material, peak H, chronopotentiometric stripping analysis, seawater samples
1. Introduction
Chronopotentiometric stripping analysis (CPSA) is an electrochemical technique introduced by Jagner and Graneli [1] for speciation of metal ions which form amalgam with mercury electrode (Zn(II), Cd(II), Pb(II), Cu(II)). Preconcentrating reduction step proceeds at negative potentials where amalgam is formed, followed by stripping step which could be performed in two different ways. Chemical oxidant (e.g. Hg(II) ions) previously added to the solution reoxidizes metals and potential in time is measured (E = f(t)). This is an example of chemical stripping. Another type of stripping mechanism is constant current stripping [2] in which constant current is applied to strip the accumulated material. CPSA is faster and more sensitive in comparison with voltammetric methods [3]. Therefore, it is successfully applied for the determination of low metal ion concentrations [4,5], metalloid ions like selenium ions [6] and organic molecules in different media, as well as in speciation analysis of metal complexes [7,8].
Organic compounds like peptides [9], proteins [3,10] and biological macromolecules like nucleic acids [11] prepared in suitable buffer solutions also show specific CPS peaks on different electrodes. Cai et al. [9] reported about CPS determination of bioactive peptides concentration based on oxidation peaks of tryptophan (Trp) and tyrosine (Tyr) on carbon paste electrode. Tyr gives oxidation peak named “Y” at Ep = 0.55 V and Trp oxidation peak “W” at Ep = 0.70 V vs. Ag/AgCl (3 M KCl). Bioactive peptides often contain Tyr residues so CPSA represents the most sensitive physico-chemical method for their determination. The stripping time increases linearly with concentration in lower concentration range, therefore subnanomolar concentrations of bombesin, neurotensin and luteinizing hormone-releasing hormone (LH-RH) could be determined. Peptide [Lys8]-vasopressin contains one disulphide bond which is reduced forming two thiol groups and one well define CPS reduction peak “S” at Ep = -0.56 V on mercury electrode appears [12]. In the same paper CPSA of peak which appears not due to the faradaic but due to the catalytic reduction was reported. That peak appears more positively on potential scale than usual hydrogen wave, preceding the reduction of sodium ions. It is named peak “H” or catalytic hydrogen or “presodium” wave and a key organic molecule is called a “presodium” catalyst [13]. Except this kind of catalyst structure, there are two more: Brdička catalysts and metal ions. Brdička catalyst is a complex of Co(II) or Ni(II) ion and S-containing organic ligand molecule [14,15]. Platinum group of metals are identified as a metal deposition catalyst [16].
In CPS catalytic reactions peak position on potential scale (Ep) and its height (dE / dt)-1 depend primarily on the molecular structure i.e. atoms or a group of atoms, their position in the molecule and surface activity. Nitrogen, sulphur, phosphorus and oxygen are the key atoms in molecules for producing catalytic peak, but only if they are suitably located in the catalyst structure. For example, nitrogen in peptide bond is not catalytically active [17]. Molecules with free electrons which attract hydrogen ions and can be easily adsorbed on the electrode surface, lower hydrogen overpotential and so hydrogen ions need less energy for reduction than without them.
Many papers deal with CPSA of commercially available isolated and/or synthesized organic molecules that are mostly of biological origin. In present paper CPSA of organic matter present in seawater samples was investigated without any pre-treatment. Namely, carbohydrates, proteins and lipids are the main groups of organic compounds found in seawater. They represent 20 % of organic matter (OM) present in natural waters and are compounds of small molecular mass characterized on molecular level in plankton and sediments [18]. Phytoplankton is the highest producer of OM in seawater's photic zone. In the dissolved fraction of OM produced by phytoplankton, carbohydrates are the most abundant group [19,20]. Significant part of seawater's polymeric material consists of polysaccharides (PS) [21,22]. Ciglenečki et al. (2003) [23] already reported about CPS detection of peak H catalyzed by sulphur atoms in polysaccharides excreted from plankton cultures exposed to anoxic conditions. Their catalytic peak H appears at Ep = -1.50 V. A similar peak is obtained in a model solution of sulphated polysaccharide, ι-carrageenan, extracted from red marine algae [24]. A more negative peak H, at around -1.7 V, was observed in seawater samples and a connection with the presence of organic matter containing N catalytic atoms was supposed. Peak potential is a well known characteristic for a qualitative analysis [25], in our case S- and N-containing OM. Therefore, in the present work catalytic activity of protein HSA which has in the structure 15 % of nitrogen atoms and of OM containing N-atoms in natural seawater was investigated in details. As the most important segment in catalysis is a contact of catalyst itself and the electrode surface [14], surface activity was examined, too. Alternating current (AC) voltammetry (out of phase mode) is widely applied for analysis of adsorption of surface active substances (SAS) on different phase boundaries by measuring the double layer capacitive current [26-28]. Therefore, this method was suitable to be used in our experiment. HSA solution was also measured with square wave (SWV) and cyclic voltammetry (CV). Because of the modified pulse excitation signal which has symmetric square shaped form, SWV is the most sensitive and the fastest among the voltammetric methods [25,29]. The concentration of (N-POM) in seawater samples was determined by CPSA and quantitatively expressed as equivalents of a model protein HSA for which a calibration line has been done in a concentration range of the expected N-POM concentrations in natural seawater. Seasonal change of N-POM was also observed and compared to SAS and DOC changes.
2. Experimental
2.1. Instrumentation
All electrochemical work was done using 663 VA Stand multimode electrode system (Metrohm, Herisau, Switzerland) and μAutolab Analyzer type III (EcoChemie, Utrecht, The Netherlands). Static mercury drop electrode (SMDE) was used as a working electrode, Ag/AgCl (3 M KCl) was a reference electrode and glassy carbon (GC) rod was an auxiliary electrode. Solution was stirred with a Teflon stirrer adjusted to 3000 turns per minute.
Dissolved organic carbon (DOC) content in seawater samples has been measured after a procedure of high temperature catalytic oxidation (HTCO) with TOC-VCPH instrument (Shimadzu, Japan).
2.2. Methods
Model protein solution and OM from natural seawater samples were electrochemically analyzed using potentiometric and voltammetric methods. Emphasis was on chronopotentiometric stripping analysis (CPSA) with constant current stripping mode, a phase-sensitive alternating current (AC) voltammetry out of phase mode (phase angle, φ = 90o), cyclic (CV) and square-wave (SWV) voltammetry.
Common measurement conditions for all electrochemical methods were: accumulation potential EA = -0.6 V and time of accumulation tA = 60 s, with stirring. Parameters being characteristic for CPSA were: constant reduction current I = -1 μA and maximum time of measurement 5 s, whereas in AC voltammetry those were: frequency f = 77 s-1, step potential Es = 0.01 V and amplitude a = 0.01 V, in SWV step potential Es = 0.01 V, amplitude a = 0.05 V and frequency f = 100 s-1, in CV step potential Es = 0.01 V and scan rate v = 1 V s-1. The whole procedure was maintained with GPES software, version 4.9.
The peak characteristics in CPSA (peak potential Ep, height (dE / dt)-1 and area - stripping time), in SWV and CV (peak potential Ep, current I and charge q) were defined after applying linear baseline. In AC voltammetric measurements the decrease of the capacitive current in the presence of SAS was measured at the potential of E = -0.6 V.
CPS and AC measurements were performed in the presence of dissolved oxygen. In contrast, the response in SWV and CV is very sensitive to the presence of oxygen, so it was important to eliminate it from solution by purging it with nitrogen for 5 minutes before each measurement. Nitrogen pressure was then maintained over the solution.
2.3. Reagents and electrolytes
A protein human serum albumin (HSA) (Sigma, Aldrich) with 15.7 % of nitrogen content, essentially fatty acid free, with molar mass of 69 kDa was a model polymer substance. One electrolyte solution was 0.55 M NaCl, corresponding to the composition of seawater but without seawater’s micro- and macroconstituents. It was prepared by diluting saturated 5.5 M NaCl solution which was made by dissolution of supra pure solid NaCl (Sigma, Aldrich). pH of 0.55 M NaCl was maintained at 8 by adding 2x10-3 M NaHCO3 to the solution. Stock solution of NaHCO3 (2x10-2M) was prepared daily from solid NaHCO3 (Sigma, Aldrich). The other electrolyte was “OM free” seawater which was prepared after filtration of seawater trough GF/F filter (Whatman) with pore size 0.7µm, UV irradiation for 24 hours and by keeping it in a mixture with charcoal for 24 hours. After that the charcoal was filtered off and “OM free” seawater was obtained. No buffer was added to the seawater electrolyte as seawater is naturally a buffer solution containing carbonate buffer system [30]. All stock solutions were prepared in MQ water from Milli-Q filter apparatus (Millipore, USA).
Seawater samples ware collected in the Northern Adriatic station 101 (Fig. 1) in 4 seasons: June and October 2008 and in January and March 2009, at surface (0.5 m depth) and bottom (~31 m depth). 25 cm3 of sample was analyzed in electrochemical cell without any pre-treatment within 24 hours after sampling.
3. Results and Discussion
3.1. Surface activity of HSA
In Fig. 2 adsorption isotherms of HSA in 0.55 M NaCl and in seawater obtained at EA = -0.6 V by AC voltammetry are presented. This is the potential of electrocapillary maximum at which the surface of mercury drop has maximum surface tension and its charge is zero. Therefore, adsorption of neutral, hydrophobic molecules or their hydrophobic parts is facilitated at this potential. By increasing the concentration of protein at the electrode, double layer capacitance and measured capacitive current were decreasing. Adsorption in both electrolytes followed the Langmuir isotherm [31]. In 0.55 M NaCl 2 mg dm-3 of HSA and in seawater 1.2 mg dm-3 HSA was needed to achieve complete surface coverage i.e. adsorption plateau (θ = 1). Dependence of electrode surface coverage (θ) on added HSA was linear in both electrolytes until above mentioned concentrations (Fig. 2B). Further addition of HSA up to 200 mg dm-3 did not lower the capacitive current.
3.2. Catalytic properties of HSA
CPSA of the model protein HSA in 0.55 M NaCl and in seawater is shown in Fig. 3 By increasing the HSA concentration, CPS peak height (dE / dt)-1 was increasing until surface coverage starts to block further catalytic effect of newly adsorbed active groups. This was starting to occur at the concentration of 0.4 mg dm-3 HSA in 0.55 M NaCl (Fig. 3A). At higher concentrations number of catalytically active N atoms in solution was increasing but they could not achieve direct contact with electrode surface because it was already covered with previously adsorbed molecules. CPSA of HSA at described conditions in seawater was possible only at concentrations lower than 0.15 mg dm-3. At higher concentrations peak H was lower or two peaks appeared. The reasons could be different orientation of catalytic groups at the electrode surface and/or different availability for catalysis. Namely, as the surface coverage increases, interactions between adsorbed molecules may be expected to become significant [32]. Higher concentrations could be analysed only at more negative value of reduction current (eq. -2 or -5 μA instead of our -1 μA) which will provide lower but reproducible peak H.
CPS signals observed in our electrolytes were result of “presodium” cathodic catalysis of electroreduction of hydrogen ions. Nitrogen atoms from HSA, bearing free electron pair, were the one that helped hydrogen ions to be reduced at the surface of mercury drop. Gaseous hydrogen was developing and peak “H” appeared. Its peak potential was situated at app. -1.70 V to -1.80 V, preceding the reduction of hydrogen ions on mercury without a catalyst. In general, if molecule has higher molecular mass it is catalytically more active [17]. Therefore, N-organic polymer like HSA was a suitable compound for producing peak H in CPSA.
The height of peak H in 0.55 M NaCl and in seawater was considerably different. For example, the height of peak H in 0.55 M NaCl for 0.1 mg dm-3 HSA was 2.6 s V-1 while in seawater for the same HSA concentration the height was 15.8 s V-1. In 0.55 M NaCl the peak potential is shifted from -1.85 V to -1.78 V, while in seawater it is shifted from -1.83 V to -1.71 V, regarding the concentrations lower than 0.15 mg dm-3 (Fig. 3B). Moving of peak potential toward positive values means that the catalytic activity is favoured i.e. the process is consuming lesser amount of energy in seawater than in NaCl. The differences in catalytic properties of HSA in 0.55 M NaCl and in seawater were due to the different adsorption of HSA i.e. different composition of the electrolytes. The ionic strength of the seawater could be modelled by using the 0.55 M NaCl solution, but besides that seawater contains other macro and microconstituents, as for e.g. it contains 0.05 mol dm-3 Mg and 0.01 mol dm-3 Ca ions. They strongly influence biogeochemical processes in seawater like adsorption [24] and complexation reactions [33]. Stronger catalytic activity was observed in seawater (Fig. 2B) because Mg and Ca cations neutralized negatively charged groups in HSA molecule and increased the molecule's affinity toward hydrophobic mercury. Therefore, sensitivity of CPSA was enhanced in the seawater.