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: strmecki@irb.hr
Other authors: plavsic@irb.hr , cosovic@irb.hr
Abstract
Catalytic properties and surface activity of nitrogen containing polymeric organic material (NPOM) 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
1
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
2
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
3
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.
4
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.
5
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
6
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
7
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.
3.3. CPSA sensitivity - comparison with SWV and CV
Catalytic signals of HSA were also recorded with voltammetric methods SWV and CV. When
compared to CPS method, these techniques have identical preconcentration step but they
differ in the stripping step. SWV and CV apply the potential scanning in the negative
direction [29] while CPS method strips material away from the electrode by applying constant
8
reduction current [2]. If the same accumulation potential and accumulation time were applied
for analysis of HSA, the results showed that constant current CPSA is a much more sensitive
than both voltammetric methods. CPSA of 0.01 mg dm-3 of HSA gave well defined peak H
already for 60 s of accumulation (Fig. 4C) while voltammetric methods did not give any peak
even for longer accumulation times i.e. for 180 s and 300 s (Fig. 4A and 4B). Similar
sensitivity results but concerning different protein solutions were reported earlier, too [3].
3.4. Calibration plot for HSA
As a result of the CPS method sensitivity, peak H height obtained in seawater electrolyte
could be utilized for quantitative determination of N-POM in seawater samples. We observed
a straight calibration line in the concentration range from 0 to 0.15 mg dm-3 of HSA (Fig. 5).
That covers the range of the peak H heights of N-POM measured in seawater samples.
Calibration plot is passing through the origin and it is described with the equation (dE / dt)-1 /
s V-1 = 154.1 x [HSA] / mg dm-3. Inset in Fig. 4 shows the increase of the peak H height and
its shift toward positive potentials with increasing HSA concentrations.
3.5. Quantitative analysis of SAS and N-POM in seawater samples from Northern
Adriatic
Organic matter in seawater samples from Northern Adriatic station 101 showed surfactant
activity i.e. it decreased capacitive current below the capacitive current value for pure
electrolyte. For longer accumulation period the decrease is higher because more SAS is
adsorbed at the electrode surface. The concentrations of SAS were expressed in equivalents of
Triton-X-100 for the accumulation time of 60 s and are given in Table 1.
CPS analysis of seawater samples gave the peak at the potential around Ep ~ -1.7 V at
the ascending part of the hydrogen evolution. That peak H is a consequence of the catalytic
activity of N-POM present in seawater samples and it was analyzed concerning different
9
accumulation times: 1 s, 30 s, 60 s, 120 s, 180 s and 300 s (Fig. 6) and stripping currents: 0.5
μA, -1 μA, -2 μA, -5 μA, -10 μA and -20 μA (not shown). As more polymeric material was
brought to the electrode, capacitive current was decreasing, peak height and area in CPSA
were increasing and peak potential was shifting toward positive values. With short
accumulation times (1 s and 30 s) peak H was not obtained probable because the amount of
material adsorbed was to low. Stripping currents higher than -2 μA and equal or lower to -0.5
μA were not suitable for measurements as either no peak H was obtained or the irregularities
in the recorded chronopotentiogram were too big. According to these findings we have
selected for analysis of seawater samples the accumulation time 60 s and the stripping current
of -1μA. Concentration of N-POM was expressed in equivalent concentration of HSA (Table
1), according to the calibration plot obtained in seawater electrolyte (Fig. 4). In samples where
no catalytic activity occurred we did not obtain any peak H (represented as zero in Table 1).
Peak H decreased upon purging of samples with nitrogen gas. For surface sample from 6 June
2008 the peak was 76 % smaller after purging a sample for 100 s with pure nitrogen gas. In
this way oxygen was purged out of the sample what caused a faster reduction of hydrogen
cations and therefore lower peak H. Purging step in CPS determination of N-POM in seawater
for analytical purposes influenced the results and should not be ignored.
From peak H height measured in the seawater samples we could not characterize NPOM on molecular basis, as for that some specific separation techniques and characterization
methods should be included. Since monomers usually do not influence strongly the capacitive
current measured at the mercury-seawater interface i.e. they have lower surface activity [34],
we could suppose that the adsorbed polymeric organic material from seawater samples was
very probably responsible for the catalytic activity. In spring/early summer and autumn in the
Northern Adriatic seawater samples polysaccharides can represent significant part of the total
carbon in seawater [21,22]. Macroaggregates found in the Northern Adriatic are characterized
10
by high C/N ratio due to their low protein content having carbohydrates as a major
component. They are mostly composed from heteropolysaccharides and polymethylene chains
and originate from phytoplankton's cell walls [35]. Besides that carbohydrates are also
recognized as N-containing molecules, having N mostly bound in the form of amine groups,
named aminosugars [36]. Nutrients and sun light are crucial factors for phytoplankton's
production/exudation of marine organic matter [37]. The Adriatic Sea is in generally an
oligotrophic semi-closed basin where phosphorus is a limiting nutrient. Its northern part,
including station 101, is seasonally greatly influenced by terrigenous nutrient input of Po
River, especially in spring and autumn because of the snow melting in Alps and frequent
rains. In the surface samples from June 2008 high concentrations of SAS and N-POM were
present, probable as a consequence of increased biological activity due to the early summer
phytoplankton bloom occurring in the photic zone. Oppositely, at the bottom layer no peak H
could be detected for 60 s accumulation and for 300 s there was only a hump (not shown),
probable because OM produced at the surface has not sunken yet to the deeper layers and the
“old” OM from previous season has been already mostly decomposed.
Concentration of N-POM expressed as eq. mg HSA dm-3 was variable through the
seasons as well as SAS and DOC having the highest values in the most productive period of
biological activity (June 2008) and the lowest value in the winter period (January 2009)
(Table 1.). Surface water was richer in OM i.e. concentration of N-POM, SAS and DOC was
higher in surface samples than in bottom samples in all seasons. To sea which part of SAS
and DOC made N-POM obtained with CPSA, we calculated N-POM/SAS and N-POM/DOC
and concluded that N-POM made a minor part of a few percent of SAS and DOC in seawater
samples.
11
4. Conclusion
The measurement of peak H position and height by constant current CPSA was a very suitable
for qualitative and quantitative determination of “presodium” catalysts as a part of OM pool
in seawater samples. Amount of N-POM was expressed as an equivalent concentration of
suitable N-containing polymer model, in our case that was protein HSA. Calibration plot of
HSA obtained in “OM free” seawater was a straight line from 0 to 0.15 mg dm-3. Peak H
strongly depends on constant stripping current applied, accumulation time as well as on
catalyst concentration, its structure and basic electrolyte. The concentrations of N-POM in
seawater samples from the Northern Adriatic were in the range from 0.0002 to 0.071 eq. mg
HSA dm-3, being higher for surface samples in the season of increased biological production
(June 2008). Concerning natural water systems, peak H represents an additional parameter for
the characterization of natural organic matter in seawater measured without any sample pretreatment. A sensitive and fast constant current CPSA is a suitable method for its analysis.
5. Acknowledgments
The financial support of the Croatian Ministry of Science, Education and Sport for the project
no. 098-098-2934-2717 under the title “The nature of the organic matter, interaction with
microconstituents and surfaces in the environment” is gratefully acknowledged. We also
thank Zdeslav Zovko for DOC measurements in seawater samples.
12
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15
Table 1. Nitrogen-containing polymeric organic material (N-POM), surface active substances (SAS), and dissolved organic carbon (DOC)
measured in seawater samples from Northern Adriatic station 101. Conditions for SAS measurements like in Fig.2 and for N-POM like in Fig. 3.
N-POM
date of
SAS
-Ep / V
(dE/dt)-1 / s V-1
eq. mg HSA dm-3
eq. mg T-X-100 dm-3
N - POM
SAS
depth / m
sampling
DOC
mg C dm-3
N - POM
DOC
6 June
0.5
1.746
9.3
0.06
0.192
0.31
1.57
0.038
2008
~31
-
0
0
0.088
0
1.04
0
26 June
0.5
1.736
11
0.071
0.363
0.19
1.85
0.038
2008
~31
0
0
0
0.136
0
1.35
0
20 October
0.5
1.791
2.8
0.018
0.251
0.072
1.01
0.018
2008
~31
1.780
0.30
0.0002
0.214
0.00091
0.80
0.00025
17 January
0.5
-
0
0
0.102
0
1.01
0
2009
~31
-
0
0
0.055
0
1.09
0
17 March
0.5
1.772
4.7
0.03
0.165
0.182
1.25
0.024
2009
~31
1.805
1.5
0.0096
0.073
0.13
0.94
0.010
16
Figure Captions
Fig. 1
Sampling station 101 in the Northern Adriatic.
Fig. 2
Adsorption isotherms of HSA in 0.55 M NaCl with added 2x10-3 M NaHCO3 (pH 8) (o) and
in precleaned seawater (●) measured with AC voltammetry with out of phase mode (= 90º)
(A) and lower HSA concentration range of isotherms (B). Conditions: EA = -0.6 V, tA = 60 s,
Es = 0.01 V, a = 0.01 V.
Fig. 3
CPSA of HSA in 0.55 M NaCl with added 2x10-3 M NaHCO3 (pH 8) (o) and in precleaned
seawater (●). Conditions for AC as in Fig. 2, conditions for CPSA: EA = -0.6 V, tA = 60 s, I =
-1 μA.
Fig. 4
CV (A), SWV (B) and CPSA (C) of 0.01 mg dm-3 of HSA in precleaned seawater,
accumulation time: 60 s (----), 180 s (- · - · -) and 300 s (- · · - · · -). Measurement without
added HSA (─). Conditions for CV: EA = -0.6 V, Es = 0.01 V, v = 1 V s-1; for SWV: EA = -0.6
V, f = 100 s-1, a = 0.05 V, Es = 0.01 V, and for CPSA as in Fig. 3.
Fig. 5
Calibration line for HSA in precleaned seawater. Inset: CPS signals of HSA, concentrations in
mg dm-3: 0) 0, 1) 0.022, 2) 0.033, 3) 0.055, 4) 0.066 and 5) 0.078. Other conditions as in Fig.
3.
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Fig. 6
Different accumulation times applied for CPSA of seawater sample from Northern Adriatic
station 101, depth 31 m, sampled on 17 March 2009. Conditions: EA = -0.6 V, I = -1 μA, tA:
1) 1 s, 2) 30 s, 3) 60 s, 4) 120 s, 5)180 s and 6) 300 s.
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