Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Bioelectrochemical methane production from CO2
    Eerten-Jansen, M.C.A.A. van - \ 2014
    Wageningen University. Promotor(en): Cees Buisman, co-promotor(en): Annemiek ter Heijne. - Wageningen : Wageningen University - ISBN 9789462570061 - 189
    methaanproductie - hernieuwbare energie - kooldioxide - elektrochemie - technieken - methane production - renewable energy - carbon dioxide - electrochemistry - techniques

    Nowadays, most of our energy and fuels are produced from fossil resources. Fossil resources are, however, finite and their use results in emissions that affect the environment and human health. For reasons of energy security and environmental sustainability, there is therefore a need to produce energy and fuels from renewable resources. However, currently several challenges need to be overcome before renewable resources can be implemented on a large scale for the production of renewable energy and fuels. At the moment, all the renewable resources can be converted into electricity. However, renewable electricity is often produced intermittently. Therefore, excess renewable electricity, when supply does not meet demand, needs to be stored not to get lost. On the other hand, fuels can currently only be produced directly from biomass. There are, however, discussions about whether sufficient biomass can be produced in a sustainable way to cover the global demand.

    A methane-producing Bioelectrochemical system (BES) is a novel technology to store excess renewable electricity in the form of methane, independent of biomass. Key principle of the methane-producing BES is the use of microorganisms as catalysts for the reduction of CO2 and electricity into methane. At the start of this thesis, the methane-producing BES was at its infancy, and for implementation of the technology a more thorough understanding of the technology was needed. Therefore, the aim of this thesis was to investigate the principles and perspectives of bioelectrochemical methane production from CO2. Focus was on the main bottlenecks limiting system’s performance.

    In Chapter 2, the performance of a flat-plate methane-producing BES that was operated for 188 days was studied. The methane production rate and energy efficiency were investigated with time to elucidate the main bottlenecks limiting system’s performance. Using water as electron donor at the anode, methane production rate was 0.12 mL CH4/m2 cathode per day and overall energy efficiency was 3.1% at -0.55 V vs. Normal Hydrogen Electrode (NHE) cathode potential during continuous operation. Analysis of the internal resistance showed that in the short term, cathode and anode losses were dominant, but with time also pH gradient and transport losses became important.

    Since the cathode energy losses were dominant, in Chapter 3, the microbial community that catalyses the reduction of CO2 into methane was studied. The microbial community was dominated by three phylotypes of methanogenic archaea, being closely related to Methanobacterium palustre and Methanobacterium aarhusense, and six phylotypes of bacteria. Besides methanogenic archaea, the bacteria seemed to be associated with methane production, producing hydrogen as intermediate. Biomass density varied greatly with part of the electrode being covered by a thick biofilm, whereas only clusters of biomass were found on other parts of the electrode.

    Based on the microbial community it seemed that methane was produced indirectly using hydrogen as electron donor. Therefore, the electron transfer mechanisms of bioelectrochemical methane production were investigated in Chapter 4. Understanding the electron transfer mechanisms could give insight in methods to steer the process towards higher rate. A mixed culture methane-producing biocathode was developed that produced 5.2 L methane/m2 cathode per day at -0.7 V vs. NHE cathode potential. To elucidate the formation of intermediates, methanogenic archaea in the biocathode were inhibited with 2-bromethanesulfonate. Methane was primarily produced indirectly using hydrogen and acetate as electron donor, whereas methane production via direct electron transfer hardly occurred.

    Besides producing methane, a BES could also be used to produce higher value organics, such as medium chain fatty acids. Currently, medium chain fatty acids are produced by fermenting (low-grade) organic biomass using an external electron donor, such as hydrogen and/or ethanol. A BES could provide the electrons in-situ, either as the electrode directly or indirectly via hydrogen. In Chapter 5, medium chain fatty acids production in a BES at -0.9 V vs. NHE cathode potential was demonstrated, without addition of an external electron mediator. Caproate (six carbon atoms), butyrate (four carbon atoms), and smaller fractions of caprylate (eight carbon atoms) were the main products formed from acetate (two carbon atoms). In-situ produced hydrogen was likely electron donor for the reduction of acetate. Electron and carbon balances revealed that 45% of the electrons in electric current and acetate, and 31% of the carbon in acetate were recovered in the formed products.

    In Chapter 6, the present performance of methane-producing BESs was evaluated. Analysis of the performances reported in literature did not reveal an increase with time. Based on the main bottlenecks that limit system’s performance as found in this thesis, methods to increase performance were discussed. Besides, we showed that our envisioned first application is to upgrade CO2 in biogas of anaerobic digestion to additional methane. Finally, the feasibility of production of higher-value organics, such as medium chain fatty acids, in BES was discussed.

    Role of protein-protein interactions on protein aggregation and emulsion flocculation
    Delahaije, R.J.B.M. - \ 2014
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga. - Wageningen : Wageningen University - ISBN 9789462570054 - 158
    methaanproductie - elektrochemie - kooldioxide - elektrolyse - microbiële brandstofcellen - duurzame energie - methane production - electrochemistry - carbon dioxide - electrolysis - microbial fuel cells - sustainable energy

    In this thesis, the effect of molecular properties on the aggregation and flocculation behaviour is studied. The aggregation behaviour was thought to be mainly affected by the structural stability of the protein. A decreased structural stability results in unfolded proteins which are more prone to aggregation. The flocculation behaviour was shown to be affected by the adsorbed amount at saturation and the adsorption rate. These parameters have been combined in a surface coverage model, which describes the stabilization of emulsions away from the iso‑electric point (pI) to be affected by excess protein in the continuous phase. In addition, a model was proposed for the prediction of the adsorbed amount at saturation. This is influenced by the protein charge and radius and system conditions (i.e. pH and ionic strength). The adsorption rate, which is a measure for the affinity of the protein towards the adsorption to the interface, was shown to increase with increasing relative exposed hydrophobicity and a decrease of the electrostatic repulsion (i.e. decrease of ionic strength or the protein charge). Close to the pI, the applicability of protein-stabilized emulsions is limited. Hence, a steric interaction was introduced to stabilize the emulsion. It was shown that glycation of the protein with a trisaccharide was sufficient to sterically stabilize the emulsions against pH-induced flocculation.

    ECA-water een mogelijk alternatief voor fungiciden : sector ontwikkelt kennis en kijkt naar mogelijkheden van toelating(interview met Jantineke Hofland-Zijlstra)
    Arkesteijn, M. ; Hofland-Zijlstra, J.D. - \ 2012
    Onder Glas 2012 (2012)2. - p. 5 - 7.
    sierteelt - ziektepreventie - gewasbescherming - elektrochemie - waterzuivering - desinfecteren - gebruikswaarde - groenten - snijbloemen - ornamental horticulture - disease prevention - plant protection - electrochemistry - water treatment - disinfestation - use value - vegetables - cut flowers
    Het gebruik van fungiciden vormt een knelpunt. Daardoor stijgt de belangstelling voor alternatieven als elektrochemisch geactiveerd water in de teelt en na-oogst. De actieve stoffen onderchlorig zuur en hypochloriet doden schimmels, bacteriën, algen en virussen in water, lucht of gewas. Dit geactiveerde water is als biocide wel toegestaan, maar een gewasbehandeling via vernevelen en spuiten niet.
    Elektrochemisch geactiveerd water in elf vragen (onderzoek van Wageningen UR Glastuinbouw geleid door J. Hofland-Zijlstra)
    Sleegers, J. ; Wageningen UR Glastuinbouw, ; Hofland-Zijlstra, J.D. - \ 2011
    Vakblad voor de Bloemisterij 66 (2011)20. - ISSN 0042-2223 - p. 34 - 35.
    sierteelt - gewasbescherming - biociden - elektrochemie - elektrische energie - waterzuivering - irrigatie - cultuurmethoden - ornamental horticulture - plant protection - biocides - electrochemistry - electrical energy - water treatment - irrigation - cultural methods
    Telers hebben de laatste tijd veel interesse in elektronisch geactiveerd water. Dit wordt door diverse leveranciers op de markt gebracht. Wat doet het en wat zijn de verschillen tussen de producten?
    Bestrijding van Botrytis in gerbera met UV-C en geactiveerd water
    Hofland-Zijlstra, J.D. ; Os, E.A. van; Hamelink, R. ; Leeuwen, G.J.L. van; Marcelis, L.F.M. - \ 2011
    ultraviolette straling - elektrochemie - botrytis - gerbera - plantenziektebestrijding - snijbloemen - desinfecteren - glastuinbouw - uv-lampen - ultraviolet radiation - electrochemistry - botrytis - gerbera - plant disease control - cut flowers - disinfestation - greenhouse horticulture - uv lamps
    Poster met onderzoeksinformatie.
    Toepassing electrochemisch geactiveerd water in de glastuinbouw
    Hofland-Zijlstra, J.D. ; Hamelink, R. ; Vries, R.S.M. de; Bruning, H. - \ 2011
    gewasbescherming - elektrochemie - desinfecteren - landbouwkundig onderzoek - glastuinbouw - plant protection - electrochemistry - disinfestation - agricultural research - greenhouse horticulture
    Poster met onderzoeksinformatie.
    Veel interesse voor ECA-water (onderzoek door Jantine Hofland-Zijlstra)
    Sleegers, J. ; Hofland-Zijlstra, J.D. - \ 2011
    Vakblad voor de Bloemisterij 66 (2011)49. - ISSN 0042-2223 - p. 33 - 33.
    sierteelt - gewasbescherming - elektrochemie - waterzuivering - chemische behandeling - desinfectie - ornamental horticulture - plant protection - electrochemistry - water treatment - chemical treatment - disinfection
    De belangstelling voor elektrochemisch geactiveerd water is groot. Dat bleek uit een grote opkomst bij de Arenasessie over dit onderwerp, vorige week donderdag in het GreenQ Improvement Center. Meer dan 80 toehoorders zaten schouder aan schouder te zweten in de luchtdicht afgesloten presentatieruimte.
    Kennisinventarisatie naar de achtergronden en toepassingen van electrochemisch geactiveerd water in de agrarische sector
    Hofland-Zijlstra, J.D. ; Vries, R.S.M. de; Bruning, H. - \ 2011
    Bleiswijk : Wageningen UR Glastuinbouw (Rapporten GTB 1087) - 38
    elektrochemie - water - toepassingen - landbouw - tuinbouw - ultrasone behandeling - verneveling - teelt onder bescherming - ziektebestrijdende teeltmaatregelen - elektrolyse - electrochemistry - water - applications - agriculture - horticulture - ultrasonic treatment - nebulization - protected cultivation - cultural control - electrolysis
    Wageningen UR Greenhouse Horticulture, with funding of Dutch Horticultural Board, has described the history and background of electrochemically activated water and explored possibilities for applications within the agricultural sector. In the Netherlands, the use of activated water as a biocide is allowed since 2009. Active ingredients of activated water are chlorine gas, hypochlorous acid and hypochlorite. Together with a high oxidation-reduction potential (ORP 750-1100 mV) there is a broad activity against bacteria, fungi, viruses, algae, protozoa and nematodes. Agricultural applications of activated water are described for seed disinfection, cleaning equipment and packing materials, removal of biofilms from pipes, disinfection of flowers, fruits and vegetables. The recent development of ultrasonic atomization of activated water created new possibilities to treat crops and harvested products against pathogens without excessive volumes of water and disinfect air from pathogens. For applications in protected crops it is desirable that the corrosive properties of the activated water should be minimized and capacities of dispensing equipment must be enlarged.
    Polyelectrolyte behaviour in solution and at interfaces
    Klein Wolterink, J. - \ 2003
    Wageningen University. Promotor(en): Martien Cohen Stuart; Willem van Riemsdijk, co-promotor(en): Luuk Koopal. - [S.I.] : S.n. - ISBN 9789058088390 - 184
    elektrolyten - grensvlak - oplossingen - elektrochemie - electrolytes - interface - solutions - electrochemistry
    Reiniging van dunne meststromen door middel van elektrodialyse
    Gastel, J. van; Vrielink, M. - \ 1996
    Praktijkonderzoek varkenshouderij 10 (1996)1. - ISSN 1382-0346 - p. 20 - 22.
    rundveedrijfmest - schoonmaken - desinfectie - elektrodialyse - elektroforese - filters - filtratie - industrie - vloeibare meststoffen - mest - varkens - elektrochemie - mestoverschotten - mestverwerking - cattle slurry - cleaning - disinfection - electrodialysis - electrophoresis - filters - filtration - industry - liquid manures - manures - pigs - electrochemistry - manure surpluses - manure treatment
    Het Praktijkonderzoek Varkenshouderij onderzocht in samenwerking met Tauw Milieu bv in Deventer de mogelijkheden voor reiniging van dunne meststromen door middel van elektrodialyse. Reiniging tot de lozingsnormen voor het riool lijkt haalbaar. De kosten voor het proces op boerderijschaal zijn echter vooralsnog te hoog.
    Electrochemical characterization of the bacterial cell surface
    Wal, A. van der - \ 1996
    Agricultural University. Promotor(en): J. Lyklema; A.J.B. Zehnder; W. Norde. - S.l. : Van der Wal - ISBN 9789054854920 - 101
    colloïden - bacteriën - celwanden - elektrokinetische potentiaal - elektrochemie - colloids - bacteria - cell walls - electrokinetic potential - electrochemistry

    Bacterial cells are ubiquitous in natural environments and also play important roles in domestic and industrial processes. They are found either suspended in the aqueous phase or attached to solid particles. The adhesion behaviour of bacteria is influenced by the physico-chemical properties of their cell surfaces, such as hydrophobicity and cell wall charge. The charge in the bacterial wall originates from carboxyl, phosphate and amino groups. The degree of dissociation of these anionic and cationic groups is determined by the pH and the activity of the surrounding electrolyte solution. Almost all bacterial cells are negatively charged at neutral pH, because the number of carboxyl and phosphate groups is generally higher than that of the amino groups. The presence of the charged cell wall groups leads to the spontaneous formation of an electrical double layer. The purpose of the present investigation is to elucidate the structure of the electrical double layer of bacterial cell surface. Such a study serves at least two goals. It allows the quantification of electrostatic interactions in the adhesion process and it contributes to gain better insight into the availability of (in)organic compounds for bacterial cells.

    The characteristics of the electrical double layer of bacterial cell surfaces have been revealed by applying a combination of experimental techniques, which include: chemical cell wall analysis, potentiometric proton titration and electrokinetic studies such as micro-electrophoresis, static conductivity and dielectric dispersion measurements.

    For the present study five Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis, have been selected. Cell walls of these bacterial strains have been isolated and were subsequently subjected to chemical analyses and proton titration studies. Both methods provide information on the number of carboxyl, phosphate and amino groups.

    The chemical analysis of isolated cell walls involves the quantitative determination of both peptidoglycan and protein content. These analyses indicate that the chemical composition of the walls of the coryneforms are very similar, but considerably different from that of Bacillus brevis. Peptidoglycan is an important cell wall constituent of the coryneform bacteria and determines about 23 to 31 % of the cell wall dry weight. The protein fractions are somewhat lower, between 7 to 14%. The cell wall structure of the Bacillus brevis strain is more complex and multi-layered. It contains a thin peptidoglycan layer, which only determines 5 % of the cell wall dry weight. On the other hand, the protein content of these walls is higher than 56%. These proteins most likely can be attributed to a so-called S(urface)-layer, which is the outermost cell wall layer.

    The surface charge density of the bacterial cells is assessed by proton titrations of isolated cell walls at different electrolyte concentrations. Rather high values, i.e. between 0.5 and 1.0 C/m 2are found at neutral pH. The absence of hysteresis in the titration curves leads to the conclusion that the charging process can be considered as reversible. It also implies that the cell wall charge is continuously in equilibrium with the surrounding electrolyte solution, at any pH and salt concentration. This observation considerably facilitates the interpretation of the titration curves, because it allows a rigorous (thermodynamic) analysis. The anionic and cationic groups in the bacterial wall could be identified and their numbers determined by representing the differential titration curves as functions of pH and cell wall charge. The carboxyl and phosphate groups are almost entirely titrated in the pH range accessible by proton titration, allowing precise estimation of their numbers. These numbers compare very well with those based on a chemical analysis of the isolated cell walls. Estimates for the number of amino groups were less accurate, because these groups are only partly titrated in the pH range were precise titration measurements are feasible. Nevertheless, it could be concluded that the number of amino groups in the bacterial wall are lower than those of the carboxyl groups.

    Information about the ionic composition of the countercharge has been obtained from Esin-Markov analysis of the titration curves and from estimates of the cell wall potential based on a Donnan-type model. The Esin-Markov analysis is purely thermodynamic and based on first principles, whereas the Donnan model requires several assumptions about the structure of the bacterial wall. Both approaches lead to the same conclusion that at salt concentrations below 0.01 M the cell wall charge is predominantly compensated by counterions, with the excluded co-ions hardly contributing to the countercharge. This observation has considerably facilitated the interpretation of the electrokinetic properties of bacterial cell suspensions.

    Electrophoresis, static conductivity and dielectric response are related (electrokinetic) techniques and therefore share common physical bases. This also implies that the physical and mathematical problems that have to be solved in order to interpret the experimental data are very similar. Analytical solutions only exist for colloidal particles for which the electrical double layer is very thin compared to the particle dimensions. Most bacterial cells are relatively large colloidal particles and therefore the largeKa theory may be of help in the evaluation of their electrokinetic properties. However, the original theories do not include surface conductance in the hydrodynamically stagnant layer. Therefore, they had to be extended to account for the finite conductivity of ions in the bacterial wall.

    Static conductivity and dielectric dispersion both show that the counterions in the bacterial wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the counterions in the bacterial wall are of the same order but somewhat lower than those in the electrolyte solution.

    Due to surface conductance the electrophoretic mobility may be strongly retarded compared to the classical Helmholtz-Smoluchowski theory, especially at low electrolyte concentrations. In 1 mM and 10 mM electrolyte solution, the Helmholtz-Smoluchowski equation underestimates the ζ-potential by approximately a factor of 2 and 1.3, respectively.

    Resolving the fundamentals of the electrochemical characteristics of bacterial cell surfaces is a key step towards a quantitative understanding of the electrostatic interactions of bacterial cells with their surroundings. The success of such an investigation depends on the state of the art of the disciplines involved. Both microbiology and colloid chemistry have the microscopically small particle as object of study. Until recently there has hardly been any exchange of scientific knowledge between these two disciplines, despite their common interest. Colloid chemists prefered to study relatively simple particles to test their basic theories and bacterial cells were considered far too complex to serve as model colloids. However, the progress that has been made during the last decades in both colloid chemistry and microbiology provide the right tools for a successful cooporation. The present study is born from such a symbiosis and shows that many physicochemical characteristics of bacterial cell surfaces are accessible with (classical) colloid chemical techniques. In fact, for testing more advanced colloid chemical theories bacteria may even be better model particles than the generally used ionorganic colloids, because of their ability to produce a homogeneous population of identical cells.

    For the time being only Gram-positive strains have been considered, because of their relatively less complex cell wall structures. Nevertheless, the techniques used may mutatis mutandis also be applied to Gram-negative cells. In fact, such a study would be highly interesting, because it would contribute to a more complete description of the composition of the electrical double layer of bacterial cell surfaces.

    Direct electrochemistry of redox proteins
    Heering, H.A. - \ 1995
    Agricultural University. Promotor(en): C. Veeger; W.R. Hagen. - S.l. : Heering - ISBN 9789054854296 - 167
    steroïden - porfyrinen - chlorofyl - flavonoïden - isoprenoïden - metabolisme - eiwitten - elektrochemie - steroids - porphyrins - chlorophyll - flavonoids - isoprenoids - metabolism - proteins - electrochemistry

    The goal of the project was to obtain more detailed insight in interactions between redox proteins and solid electrodes and the mechanisms of electron transfer. In addition to this, the influence of the protein environment on the redox properties of the active site and the possible influence of the electrode/promoter system on these properties have been considered. Because redox enzymes do not often give an unambiguous and reversible electrochemical response (if at all), electron transferring proteins have been studied. The FMN containing flavodoxins have been used as model systems for flavin enzymes such as glucose oxidase. A series of high potential iron-sulfur proteins (HiPIPs) can be regarded as models for proteins containing [4Fe-4S] clusters. The HiPlPs are also of interest because of the high oxidation state of the cluster; the sequences are known, and the three-dimensional structures of some HiPIPs are known.

    In Chapter 2 the electrochemical behaviour of flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been characterized by staircase cyclic voltammetry (SCV) and differential pulse voltammetry (DPV). Fully oxidized flavodoxin at the bare glassy carbon electrode gave one redox couple at a potential of -218 mV (NHE) at pH=7.0 with an SCV peak current proportional to the scan rate. This response is caused by FMN, dissociated from the protein and adsorbed onto the electrode. The midpoint potential and the p K of 6.5 are equal to the values measured with free FMN in solution. When the cationic promoter neomycin was added one additional and diffusion controlled response was observed. This positively charged aminoglycoside is believed to form a flexible bridge between the negative charges on the surface of both the protein and the electrode. The midpoint potential of the observed redox couple is -413 mV (NHE) at pH 7.0 with a redox-linked pK for the reduced form of 4.8. The temperature dependence is -1.86 mV/K, yielding ΔS°=-179 J.mol -1.K -1and ΔH°=-12.4 kJ/mol. This response is believed to be the semiquinone/hydroquinone transition. Although the starting material was 100% quinone, no response was observed around the midpoint potential of the quinone to semiquinone reduction of -113 mV (NHE) at pH 7.0, determined in an EPR-monitored titration with dithionite. Digital simulation shows that the peak currents of the second reduction couple approach a maximum value after a few cycles if comproportionation of fully reduced and fully oxidized flavodoxin occurs in solution and a small amount of semiquinone is either present initially or is generated by mediation of electrode-bound FMN. In the latter case the observed increase of the peak height can be fitted with a Butler-Volmer type heterogeneous electron transfer rate between adsorbed FMN and flavodoxin of 6.3-10 -6m/s. This anomalous behaviour might have implications for the interpretation of electrochemistry on flavin enzymes like glucose oxidase. The observed peak is not per se the expected protein response or the expected (first) reduction step. The development of a catalytic current when substrate is added is no prove for direct interaction between the protein and the electrode, but can also be accomplished by electron transfer mediated by a small amount of free flavin.

    In Chapter 3 the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478) are reported. Since wild-type flavodoxin dimerizes via disulphide bond formation between cysteine 69 residues, Cys69 has been replaced by an alanine as well as a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promoter neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode, similar to the observations with D. vulgaris flavodoxin. However, addition of dithiotreitol proved to be necessary for the stabilization of the wild-type flavodoxin response. Dithiotreitol probably prevents dimerization of the protein by formation of cystine bridges. EPR- monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirm the cyclic voltammetry data. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described with a simple one-p Kred model. Instead, the presence of at least two redox-linked protonation sites is suggested: p Kred,1 = 5.39 ± 0.08, p Kox= 7 .29 ± 0.14 and p Kred,2 = 7.84 ± 0.14 with Em7 = -459 ± 4 mV and a constant potential at high pH of -485 ± 4 mV. The dependence of the semiquinone/hydroquinone potential on temperature is -0.52 ± 0.06 mV/K, yielding Δ H ° = 28.6 ± 1.5 kJ/mol. and ΔS° = -50 ± 6.2 J.mol -1.K -1. No significant differences in redox properties of wild- type, C69A and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of FMN by either an alanine or a serine residue does not have a measurable influence on the structure of the protein.

    In Chapter 4 a theoretical solution of the concentration distribution in long optical path length thin layer spectroelectrochernical cells is given by a convergent infinite summation of terms. At short times a large number of terms is required to obtain a good approximation. Alternatively, on a timescale at which the boundary of the diffusion layer has not reached the cell wall opposite to the electrode the concentration profile of the thin layer cell is equal to the profile of a semi-infinite bulk electrochemical cell. This profile is described by an error function, for which no analytical solution is available. A new three-parameter exponential approximation for this error function is presented with an accuracy better than 0.05% for all positive values of x . When the diffusion layer boundary reaches the cell wall the semi-infinite bulk model is no longer valid but then the slope of the profile has become small enough to be approximated by only the first five terms of the summation. When the composition of the bulk solution is measured by a light beam passing at grazing incidence over the electrode surface, the absorbance can be calculated from the concentration distribution by integration of the transmittance perpendicular to the light beam.

    In Chapter 5 the validity of the mechanism proposed in Chapter 2 for the flavodoxin response has been verified by measuring the absorbance of the semiquinone form of D. vulgaris flavodoxin during cyclic voltammetry. A long optical path length thin layer electrochemical cell (LOPTLC) was used with a layer width of 0.2 mm. Despite the non-ideal behaviour of this cell, the resulting "cyclic voltabsorptomograms" clearly show the proposed formation of semiquinone by FMN-mediated electron transfer, and comproportionation of flavodoxin in solution occurs. Simulated voltabsorptomograms qualitatively confirm this, although the observed reoxidation of flavodoxin semiquinone at low scan rates is not predicted by the Butler-Volmer model of Chapter 2.

    In Chapter 6 the High Potential Iron-Sulfur Proteins (HiPIPs) from Ectothiorhodospira vacuolata (iso-1 and iso-2), Chromatium vinosum , Rhodocyclus gelatinosus , Rhodocyclus tenuis (strain 2761), Rhodopila globiformis and the large (multimer) HiPIP (iso-2) from Rhodospirillum salinarum have been investigated by direct electrochemistry. Using a glassy carbon electrode with a negatively charged surface, direct, unpromoted electrochemistry was possible with the positively charged HiPlPs. With the negatively charged HiPIPs the positively charged and flexible bridging promoter poly-L-lysine was required. The stability of the response could be improved by morpholin in combination with the negatively charged proteins and by monomeric amino acids or 4,4'-dipyridyl with the positively charged HiPlPs. These 'stabilizers' apparently prevent the blocking of the electrode by denatured protein during electrochemistry. The redox potential of 500 mV found for the large HiPIP from R. salinarum is the highest HiPIP potential reported. The presence of histidines in the sequence does not per se predict a pH-dependent redox potential. Only C. vinosum and R. gelatinosus HiPIPs show a weak but significant pH dependence with a difference of 35 mV between the low and the high pH form and maximum slopes of about -20 mV/unit. Either the coupling of electron and proton transfer is indirect ('allosteric') or p Kox is only 0.6 units lower than p Kred . In the latter case an apparent dielectric constant of 48 can be calculated. The dependence of the midpoint potential on ionic strength cannot be explained by the Debye-Mickel theory alone because the linearity exceeds the limiting concentration, the slopes are much smaller than predicted by this theory (0 to -28 mV/vM) and no positive slopes are observed. Combination of the sequences, the optical spectra, the overall charges and the redox thermodynamics suggests the existence of two major groups of HiPlPs. One group consists of Chromatium -like HiPIPs with redox potentials between 300 and 350 mV, modulated only by the solvation of the cluster but not by the overall charge of the protein. The second group is formed by Ectothiorhodospira -like HiPIPs with potentials between 50 and 500 mV, largely dependent on the overall charge of the peptide and also modulated by cluster solvation. From the slope of 25 mV per unit charge an apparent dielectric constant of 84 is calculated.

    In Chapter 7 the reversible 2 x 1 e-reduction of the cubane cluster from oxidized to reduced to super-reduced: [4Fe-4S] 3+= [4Fe_4S] 2+= [4Fe-4S] l+has been studied in the HiPlPs of Chapter 6. Super-reduction to the 1+ state was not observed in any of these seven HiPlPs tested during cyclic voltammetry (down to -0.95 Volt). However, equilibration at low potential (pH 7.5) of Rhodopila globiformis HiPIP yielded a transient peak around -0.47 V due to the oxidation of super-reduced HiPIP adsorbed at the electrode. The peak area depends on the equilibration potential according to a one-electron Nernst curve with a half-wave potential at - 0.91 V. Reduction of R.globiformis HiPIP with titanium(III)citrate at pH 9.5 is very slow (pseudo first-order half-life of 23 min. with hundred-fold excess Ti(III)) but is reversible, and the EPR spectrum with g -values of 2.04 and 1.92 is similar to that of reduced [4Fe-4S] l+ferredoxins. Chemical or electrochemical reoxidation of the super-reduced form resulted in an EPR spectrum with g ¦¦ = 2.12 and g- = 2.03, i.e. identical to that of oxidized HiPIP. From the equilibrium concentration of super-reduced HiPIP at low concentration of Ti(III) a reduction potential of -0.64 V can be estimated. Super-reduction of the large HiPIP (iso-2) from Rhodospirillum salinarum is also possible with Ti(III) ( gz = 2.05) but the superreduced state is unstable. No super-reduction with Ti(III) was observed for the other HiPIPs. The difference between the electrochemically observed reduction potential and oxidation potential is explained by a fast and reversible conformational change upon super-reduction. The rate of super-reduction with Ti(III) is limited by the small amount (0.1 %) of the HiPIP in the 2+ state with the super-reduced conformation.

    It can be concluded that the interaction of redox enzymes with the glassy carbon electrode is determined primarily by the charge of the protein and of the electrode surface. With positively charged proteins no promoter is required to obtain direct electron transfer at the negatively charged electrode surface. A positively charged promoter must be added to obtain a response with negatively charged proteins. A flexible promoter that can adjust its shape to fit both the protein surface and the electrode surface gives the best results. However, the response usually deteriorates in time. This is probably caused by denaturing of the protein on the electrode surface. Van der Waals forces and hydrophobic interactions probably play an important role in this process. The electrode thereby becomes gradually less accessible for electron transfer. A good promoter therefore not only forms a flexible bridge that compensates the electrostatic repulsion, but must also protect the protein from the hydrophobic patches on the electrode. Neomycin apparently has this double- function as promoter for flavodoxins. However, the HiPIP studies show that although poly-L-lysine promotes the response of the negatively charged proteins, the response is not stable. A separate "stabilizer" can be added to improve the voltammetry of both positively and negatively charged HiPlPs. The wild-type Azotobacter vinelandii flavodoxin 11 is a special case where dithiotreitol stabilizes the response by preventing the formation of dimers in which the FMN is no longer accessible to the electrode.

    The redox potential of a protein is not always influenced by the charges of the peptide. This is true for both the permanent charges and for the pH-dependent charges. The HiPIP studies show that the distance between the charge and the redox-center, the dielectrics of the peptide in between, and the exposure of the charged groups to water are important factors. The dependence of the redox potentials on pH, measured with electrochemistry, do not always agree with the results of bulk-titrations. This can just be an indication that the electrochemical measurements have a much better accuracy, but some influence of the electrode surface and/or the promoter on the protein structure cannot be excluded. It is therefore important to compare the electrochemical data with the results of independent spectroscopic redox-titrations. Simultaneous spectroscopic measurements of the solution during the voltammetric experiments can give useful additional information for the deconvolution of coupled homogeneous reactions.

    Electrochemical metal speciation in colloidal dispersions
    Wonders, J.H.A.M. - \ 1995
    Agricultural University. Promotor(en): J. Lyklema; H.P. van Leeuwen. - S.l. : Wonders - ISBN 9789054854708 - 88
    chemische speciatie - zware metalen - colloïden - elektrochemie - chemical speciation - heavy metals - colloids - electrochemistry

    The term "heavy metals" is connected with toxicity. They form strong complexes with enzymes, other proteins and DNA in living organisms, which causes dysfunctioning and hence poisoning. In combination with the uptake mechanism of the organism, speciation of heavy metal determines the bio-availability of heavy metals. In the environment, heavy metals are complexed by soil particles or molecules of organic and inorganic origin. This thesis deals with the speciation and the binding characteristics of heavy metals. Since complexation of heavy metals with soil particles is far too complex because of the wide range of different particles, this investigation is restricted to binding to a model system. The model system consists of polystyrene latices with and without a hydrophilic polymer shell. The surfaces of these latices contain negatively charged surface (shell) groups which can act as metaI-complexing agents. The binding can be investigated using various types of voltammetric techniques (Chapter I). To study metal binding, we first determined the amounts and types of surface groups present on the latices using potentiometry and conductometry (Chapter II). The polystyrene latex without shell showed a very high density of mainly weak, carboxylic groups on the surface. The surfaces (and shell) of the core-shell latices consist of a fraction strong acids (sulphonics) and a fraction of weak acids (carboxylics). Their shells and surfaces contain a lower total amount of groups than the polystyrene latex without shell. All conductometric results are qualitatively in agreement with those obtained by potentiometry, although the conductometric data appear to be more accurate. Potentiometry using potassium hydroxide, followed by a titration using nitric acid, was performed on one core-shell latex, indicating reversibility. During the titration with KOH, surface groups in the shell migrate to the surface. This effect is reversible. For one core-shell latex, potentiometric studies were carried out at different concentrations of supporting electrolyte (potassium nitrate). As expected, the pH increases more the lower the ionic strength during a titration. The total amount of titratable surface groups increases with higher concentration of supporting salt.

    As a following step, the metal complexes formed were characterized (Chapter III). voltammetric experiments, such as Cottrell type experiments, with all core-shell latices studied, show the formation of labile zinc(II) and cadmium(II) ion complexes at very low metal-to-site ratios in the time scale of pulse voltammetry. This means that the residence time of the metal ion in the complex form is very small compared to the pulse time. The application of the voltammetric model of de Jong et al. for dissolved complexes is succesfully used for the analysis of the binding of metal ions by colloidal particles. At a decreased metal to ligand ratio, the complexes formed were still labile, but their stability constants were slightly higher. Perhaps there is a minority of strong complexing surface groups, due to clustering or impurities in the shell, resulting in different affinities for metal ions. The metal/carboxylate surface complexes of the highly charged latex lose lability at high degree of dissociation. Also, stability constants obtained from the normalized current diverged from those obtained from the potential shift, with higher stability constants for the latter one. Some aspects of this discrepancy are discussed. The calculation of the kinetics of the lead-carboxylate complexes using the lability criterion of de Jong et al . shows that these complexes are marginally labile.

    Chapter IV deals with the characterization of surface groups by voltammetric titration, which is more complex than often assumed. This chapter tackles some of the methodological pitfalls which can be easily overlooked. Further, we estimated the amount of cadmium-complexing surface groups of some latices. The (complete) titration curves for all latices are regularly shaped. At the very onset of the titration curves complexes with larger binding constants were formed. This is probably due to the heterogeneity in surface groups described above. A procedure in which a regression line is computed using the diffusion coefficient of the latex metal complexes, can be used in the analysis. This procedure also provides one of the checks whether or not a metal complex is labile. The cadmium(II)-complexing capacity of the latices increases parallel to the fraction of carboxylic groups. Assuming a 1:2 binding ratio, roughly 30% of the sulphonate groups and 80% of the carboxylate groups bind cadmium(II) It seems that charge compensation plays a major role. Since the complexes formed by the polystyrene latex with a very high density of carboxylic groups only are not labile, the data for this latex were treated as if its surface sites would form inert complexes. An impression about the error of this treatment can be given; it seems rather small, just a few percent, due to the low diffusion coefficient of the latex particles.

    On the basis of potentiometric titrations at varied supporting electrolyte concentrations, we applied Donnan and Donnan-derived models by Ohshima and Kondo to describe the proton binding using the potential in the shell of a latex in Chapter V. In addition, the cadmium-binding properties of a core-shell type of latex were determined using differential pulse polarography. The assumptions in the shell potential model used are: the shell has a constant thickness independent of the ionic strength, the relative dielectric permittivity coefficient is 80, the degree of dissociation is constant over the shell and the site distribution is homogeneous. These assumptions did not affect the description of proton binding to a core-shell latex. Donnan's approach describes reasonably well the proton binding on the surface groups of the core-shell latex coded AOY5. Ohshima's model refines this description, by taking a Poisson-Boltzmann distribution of ions near and in the shell into account. This is an improvement. It seems that the potential correction based on the (indifferent) salt concentration is a major parameter for the binding of protons. The logarithm of the intrinsic cadmium binding constant (extrapolated to a shell charge of zero) is 1.0-1.2 for the carboxylic groups, comparable to corresponding bulk values for various organic cadmium-carboxyl complexes.

    Characterization of redox proteins using electrochemical methods
    Verhagen, M. - \ 1995
    Agricultural University. Promotor(en): C. Veeger; W.R. Hagen. - S.l. : Verhagen - ISBN 9789054853800 - 145
    oxidoreductasen - cytochromen - elektrochemie - oxidoreductases - cytochromes - electrochemistry

    The use of electrochemical techniques in combination with proteins started approximately a decade ago and has since then developed into a powerfull technique for the study of small redox proteins. In addition to the determination of redox potentials, electrochemistry can be used to obtain information about the kinetics of electron transfer between proteins and about the dynamic behaviour of redox cofactors in proteins. This thesis describes the results of a study, initiated to get a better insight in the conditions necessary to obtain electron transfer between solid state electrodes and proteins.

    Flavin Adenine Dinucleotide (FAD) is the subject of chapter 2. The electrochemical behavior of this cofactor, which is present in some flavoproteins, appeared to be dependent on its solution concentration. At concentrations of 1 μM the voltammetry showed all the characteristics of a species adsorbed to the surface. At a thousandfold higher concentration the voltammetry became completely diffusion controlled. From experiments at intermediate concentrations it was concluded that part of the FAD molecules adsorb to the electrode. Furthermore, it was shown that electron transfer between the molecules in solution and the electrode can only take place through the adsorbed molecules, which act as mediators. A comparison with results obtained with a 2 [4Fe-4S] ferredoxin from Megasphaera elsdenii suggested that, under certain conditions, a similar mechanism of selfmediation can be valid for proteins.

    The results of a study of cytochrome c 553 from D. vulgaris (H) are presented in chapter 3. The cytochrome was characterized by cyclic voltarnmetry and the same technique was used to determine the rate of electron transfer between the cytochrome and the Fe-hydrogenase from the same organism. The results indicated that the cytochrome was a physiologically competent redox partner dependent on the in vivo function of the hydrogenase. Since the function of the hydrogenase is still an issue of debate it is not known whether this new electron transfer pathway has physiological relevance.

    The reinvestigation of the protein desulfoferrodoxin from D. vulgaris (H) is described in chapter 4. This protein was reported to contain two iron atoms one of which was coordinated by four cysteine residues in a distorted tetrahedron. By comparison with model compounds using EPR spectroscopy and by using cyclic voltarnmetry at different pH values it was shown that this is very unlikely. Instead it is proposed that the iron atom is coordinated in a pentagonal bipyramid (surrounded by 5 ligands in a plane and 2 ligands perpendicular to and on both sides of this plane). Furthermore the controversy about the protein having a mixed N-terminus was elucidated and it was established that the protein was a homodimer instead of the reported monomer.

    The conditions necessary for the use of direct electrochemistry to study small redox proteins become more and more established. The application of this technique to enzymes is, however, not straightforward, The reason for this is not clear, but one possibility is that a large enzyme adsorbs more easily to the electrode than a small protein. Another possible explanation is that the redox cofactors in enzymes are shielded more by the protein matrix. In order to circumvent this latter problem we tried to establish conditions for the electron transfer between cytochrome P- 450 from P. putida and glassy carbon electrodes. This bacterial cytochrome P-450 has a ferredoxin as a physiological electron donor and has therefore a docking place where the electrons can enter the enzyme. When using the right conditions it should be possible to let the electrode be the "substrate" for the enzyme. Unfortunately the enzyme adsorbed to the electrode and the obtained value for the potential was much more positive than the literature value. An EPR redox titration of the cytochrome P-450 indicates that the literature value needs a correction. However, there still remains a difference between the value obtained from the titration in homogeneous solution and the value determined electrochemically.

    Recent reports about electrochemical characterization of superoxide dismutase from bovine erythrocytes incited the study described in chapter 6. The conditions used in the reported electrochemical experiments were rather extreme i.e. low pH. EPR monitored redox titrations of the enzyme at different pH values indicated that the oxidation and reduction at low pH values is not reversible. Furthermore, it was found that the reported potentials at pH 7.0 needed to be corrected. A redox titration was also performed with the iron enzyme from E. coli as a comparison with the copper zinc containing enzyme. After reduction, however, it was not possible to reoxidize the enzyme again indicating that the redox reaction is not reversible. This can explain the huge differences in potentials reported so far in the literature.

    The use of a redox active promotor can give some insight in its mechanism of action. The lanthanide europium proved to be a potent promotor of rubredoxin. The latter is a small purple redox protein containg a single iron coordinated to 4 cysteine residues. At high pH values the reduction and oxidation of rubredoxin is readily obtained despite the fact that the europium ion does not show any reduction or oxidation anymore. This is not consistent with the models used to explain the promotor function of cations. These models all assume that the cation provides charge compensation and sandwiches both between the protein and the electrode as well as between different protein molecules. The results from this study are presented in chapter 7.

    A great advantage of electrochemistry using glassy carbon electrodes is that it is possible to vary the potential between approximately -1 V and +0.8 V. This makes it possible to study redox couples with potentials more negative than the commonly used chemical reductants like dithionite or titanium citrate. This led to the discovery of the superreduction of the Rieske cluster in the soluble fragment of the bc1 complex of beef heart as described in chapter 8. This protein contains an [2Fe- 2S] cluster with a redox potential of + 312 mV versus SHE. At low potential (-840 mV versus SHE) it is possible to reduce this cluster with a second electron. The physiological relevance of this superreduced state is not clear but its characterization can give insight in the mechanism of multiple electron transfer by iron sulfur clusters.

    The final two chapters are used to describe the biochemical and spectroscopic characterization of two proteins from D. vulgaris . Adenylyl sulphate reductase (AdoPSO 4 reductase) is an enzyme which is involved in the sulfate respiration of the bacterium. It reduces the activated sulfate (AdoPSO 4 ) to AMP and sulfite. Literature reports indicated that the protein contained one FAD and two [4Fe-4S] clusters. The presence of two clusters was based on the observation of a complicated EPR spectrum which indicates interaction between two paramagnetic centers. In our studies however this "interaction" spectrum only appeared when the enzyme solution was at low ionic strength. Upon raising the ionic strength with an inert salt like NaCl the complicated EPR spectrum changed into a spectrum of a single S-1/2 species. This indicated that the interaction between the paramagnetic centers was intermolecular rather than intramolecular. This observation led us to propose that AdoPSO 4 reductase contains one FAD and one Fe-S cluster. Since the average metal analysis showed the presence of 6 iron atoms and 5 acid labile sulfur atoms it was proposed that the Fe-S cluster may have an iron content greater than 4.

    Chapter 10 describes the results of a study of high molecular weight cytochrome c . This protein resides in the periplasmic space of D. vulgaris and contains sixteen hemes. Its function is up till now unknown. In previous reports midpoint potentials were reported for the different hemes based on single scan differential pulse voltammetry. These values might be erroneous due to the absence of reversibility. Indeed an equilibrium redox titration monitored by EPR indicated that the reported values were incorrect. Furthermore, it was not possible to reproduce the reported voltammograms. This confirmed our observation that the electrochemistry of large proteins or enzymes is often difficult to interpret and difficult to reproduce. It is also a good example of how important it is to check whether or not reversibility applies during electrochemical experiments.

    Electrochemical metal speciation in natural and model polyelectrolyte systems
    Hoop, M.A.G.T. van den - \ 1994
    Agricultural University. Promotor(en): J. Lyklema, co-promotor(en): H.P. van Leeuwen. - S.l. : Van den Hoop - ISBN 9789054852179 - 131
    metalen - elektrolyten - chemische speciatie - elektrochemie - dubbelzouten - metals - electrolytes - chemical speciation - electrochemistry - double salts

    The purpose of the research described in this thesis was to examine the applicability of electro-analytical techniques in obtaining information on the speciation of metals, i.e. their distribution over different physico-chemical forms, in aquatic systems containing charged macromolecules. In chapter 1 a general introduction is given to (i) metal speciation in aquatic systems, (ii) (bio)polyelectrolytes and their counterion distributions and (iii) electrochemical methods emphasizing their apllication to metal speciation.

    Chapter 2 deals with the conductometric: measurement of counterion association with macromolecules. First, we have surveyed theoretical developments concerning ion association for purely electrostatic interaction and as reflected in the conductivities of polyelectrolyte solutions. It will be shown that for the salt free case, the distribution of monovalent counterions can be obtained from plots of the molar conductivity of the polyelectrolyte solution versus the molar conductivity of the monovalent counterion, so-called Eisenberg plots. Experimental results for various alkali polymethacrylate concentrations show that the fraction of conductometrically- free monovalent counterions is in close agreement with theoretical predictions, which are based on a two-state appoach. Furthermore, for linear polyelectrolytes a recently proposed model for the case of counterion condensation in systems with ionic mixtures is presented. Finally, the treatment of conductometric data for polyelectrolyte solutions with either one type of counterion or mixtures of two types of counterions in terms of free and bound fractions is discussed.

    In chapter 3 we describe a voltammetric methodology for the analysis of labile homogeneous heavy metal-ligand complexes in terms of a stability K . The method takes into account the difference between the diffusion coefficients of the free and bound metal. Since the relationship between voltammetric current and mass transport properties under stripping voltammetric conditions is not yet well esthablished, we propose a relationship between the experimentally obtained current and the mean diffusion coefficient of the metal-complex system. A sensitivity analysis of this expression for different parameters, such as the stability and the ratio of the diffusion coefficients of the bound and free metal is performed.

    Natural complexing agents are often heterogeneous with regard to their affinity to metal ions. Therefore, we will discuss the evaluation of the heterogeneity of these complexes from voltammetric data for various metal-to-ligand ratios. For the case of a large excess of ligand over the metal atom concentration, the stability of the metal-complex system may be obtained independently from the potential shift. For this an equation is given similar to the classical one derived by DeFord and Hume. Finally, we present an experimental procedure based on adding ligand to the solution of the metal and measuring its voltammetric characteristics. The procedure takes into account (i) possible adsorption of metal ions to elements of the equipement and (ii) measuring all protolysis of the polyacids involved.

    The characteristic features of applying the two electrochemical techniques conductometry and voltammetry to the study of ion binding by polyelectrolytes are discussed and compared in chapter 4. Analysis of data on K +/Zn(II)/polyacrylate and K +/Zn(II)/polymethacrylate systems illustrates a certain complementary of the two methodologies. Conductometry primarily measures the Zn(II)/K +exchange ratio. Voltammetry measures the Zn(II)/polyion binding strength; its dependence on the (excess) K +concentration also yields information on the Zn(II)/K +exchange ratio. The different results seem to be fairly coherent.

    Experimental conductometric and voltammetric speciation data of metal-synthetic polyacid systems are presented and discussed in chapter 5. The competitive binding of monovalent and divalent counterions has been studied by the conductometric procedure described in chapter 2 for aqueous solutions of alkali metal polymethacrylates in the presence of Ca(NO 3 ) 2 and Mg(NO 3 ) 2 . The experimentally obtained fractions of conductometrically free counterions are compared with theoretical values computed according to a new thermodynamic model described in the same chapter. For the systems studied, the fractions of free monovalent and divalent counterions can be fairly well described by the theory. In fact, the results support the assumption that under the present conditions the conductometrically obtained distribution parameters f 1 and f 2 approximate the equilibrium fractions of free monovalent and divalent counterions. The experimentally obtained M +/M 2+exchange ratios agree well with the theoretical ones. Similar experiments have been performed for the Zn(II)/polyacrylate and Zn(II)/polymethacrylate systems. It seems that, compared to Ca 2+and Mg 2+ions, the Zn(II)-ions are bound more strongly. This could be due to some specific binding of Zn(II)-ions. Since the theoretical model does not incorporate this mechanisme, the experimental results do not agree well with the theoretical ones.

    Furthermore, chapter 5 collects the results of a systematic study of the stripping voltammetric behaviour of Zn(II)- and Cd(II)-ions in polyacrylate and polymethacrylate solutions. All metal- ligand complexes involved apprear to be voltammetrically labile over the whole range of metal-to- ligand ratios under the various experimental conditions employed. Hence, the voltammetric data could be analyzed in terms of a stability K according to the methodology presented in chapter 3. The first set of experiments is concerned with the influence of the molar mass of the polyacrylate anion on the stability. Analysis of the data in terms of a mean diffusion coefficient, which decreases with increasing molecular mass, yields a consistent picture with molar mass- independent complex stabilities. The speciation of Zn(II) in such a polyelectrolyte system varies with the concentration of carboxylate groups, but it is invariant with the polyionic molar mass. Secondly, the competition between monovalent (K +) and divalent (Zn(II) and Cd(II)) counterions has been investigated by varying the concentration of electroinactive supporting electrolyte. The results show that the stability of the heavy metal/polymethacrylate complex decreases with increasing KNO 3 concentration. This effect is largely due to the reduction of the electrostatic component of the metal/polyanion interaction, which is generally the case for polyelectrolytes with high charge densities. For the Zn(II)/polymethacrylate system, a comparison with conductometric data representing the competitive behaviour of monovalent and divalent counterions has been made in chapter 4. The influence of the polyelectrolyte charge density of the polymethacrylic acid on the stability K has been studied by varying the degree of neutralization of the polyanion. For the Zn(II)/PMA complexes, the stability increases approximately linearly with increasing degree of neutralization, i.e. with increasing polyionic charge density. This is in accordance with the general polyelectrolytic feature that counterion binding is stronger with higher polyionic charge density. Finally, for later comparison with natural complexing agents, the chemical homogeneity of the macromolecules involved has been verified by varying the total metal ion concentration for a given polyelectrolyte concentration. The results indeed confirm that the Zn(II)/polymethacrylate and Cd(II)/polymethacrylate complexes have a homogeneous energy distribution. This is in line with expectation, since these macromolecules consist of only one repeating chemical binding site, i.e. the carboxylate group.

    Chapter 6 deals with the pretreatment and characterization of humic material. The pretreatment procedure is used to purify the humic material in such a way that (i) the molecules are soluble under the experimental conditions employed in chapter 7, (ii) the amount of impurities is minimized and (iii) the resulting humic material is transferred into the acid form. Furthermore, a fractionation method based on the solubility of the humic substances is described. The humic material is characterized in terms of (i) the amount of chargeable groups by means of conductometric titration and (ii) molar mass distribution by flow field-flow-fractionation. It will be shown that although the fractionation by varying pH results in samples with different molar masses, the separation is far from ideal.

    As was done with the synthetic polyacids, experiments have been performed for natural occuring polyelectrolytes. Conductometric and voltammetric results forvarious metal humic acid systems are presented in chapter 7. Solutions of humic acids were conductometrically titrated with potassium, sodium, lithium, calcium and barium hydroxide solutions. The results have been analyzed in terms of fractions of free and bound metal. The conductance properties of humic acids are basically different from those of a linear polyelectrolyte such as polymethacrylate. A marked difference was observed between the shapes of the curves for alkali metal hydroxides and those for alkaline earth metal hydroxides. It appears that monovalent cations are hardly bound by the humate polyion, whereas divalent counterions show a strong interaction. The latter feature may be fruitfully utilized in quantitative analysis.

    The association of the heavy metals zinc(II) and cadmium(II) with humic acid samples has been furthere studied by differential pulse anodic stripping voltammetry (i) for various concentrations of supporting electrolyte (KNO 3 and Ca(NO 3 ) 2 ), (ii) for different degrees of neutralization of the humate polyion, (iii) for different metal-to-ligand ratios and (iv) for different fractions of the humic acid. Under the experimental conditions employed, all heavy metal/humate complexes have been found to be voltammetrically labile over the whole range of metal-to-ligand ratios. Hence, the stability (K) of the complex could be computed taking into account the difference between the diffusion coefficients of the free and bound metal. The dependence of K on the concentration of 1-1 electrolyte (KNO 3 ) is of comparable extent for various metal-humate complexes, but significantly smaller than in the case of the highly charged linear polyelectrolyte polymethacrylic acid. For the humic acid systems, it has been concluded that the relatively weak dependency of K on the salt concentration mainly reflects screening effects. The influence of the concentration of 2-1 electrolyte (Ca(NO 3 ) 2 ) on the stability of the heavy metal/humate complex is more pronounced than for the corresponding case of 1-1 electrolyte. By taking into account the association of calcium with the humate polyion, the stability of the heavy metal/humate complex was found to be more or less constant over the range of Ca(NO 3 ) 2 concentrations studied and comparable to the stability of the corresponding complex in the absence of calcium.

    The stability of the heavy metal/humate complex has been found to increase with increasing degree of neutralization, i.e. with increasing charge density of the humate polyion. It seemed that the increase of K is less pronounced for higher values of α n . This observation could not be interpreted from an electrostatic point of view, and is in fact a further indication that the binding of heavy metals with the humate polyion is mainly governed by the chemical characteristics of the humic acid. The chemical heterogeneity of the humic acids was investigated by varying the metal-to-ligand ratio for different total concentrations of the heavy metals but in a range of comparable ligand concentrations. The results show that the stability K of the heavy metal/humate complex decreases with increasing total metal ion concentration, reflecting a certain chemical heterogeneity of the humic acid. For various heavy metal/fractionated humate complexes, the stability K was found to be comparable to the K value for the corresponding unfractionated humic acid system. This means that the distribution of functional groups is more or less the same for different molar masses of the humic acid.

    For the present metal/humate complexes, the general conclusion is that the distribution of counterions over the free and bound states is mainly governed by the chemical heterogeneity of the humate polyion.

    Electrochemical and structural characterization of self-assembled thiol monolayers on gold
    Sondag-Huethorst, J.A.M. - \ 1994
    Agricultural University. Promotor(en): G.J. Fleer, co-promotor(en): L.G.J. Fokkink. - S.l. : [s.n.] - ISBN 9789074445160 - 204
    oppervlakten - grensvlak - goud - organische zwavelverbindingen - elektrochemie - oppervlakteverschijnselen - surfaces - interface - gold - organic sulfur compounds - electrochemistry - surface phenomena

    Self-assembled alkanethiol monolayers on gold are used as model systems in a fundamental study of the potential-dependent wetting and of the galvanic metal deposition. For using such monolayers as model systems, well-defined and ordered monolayers are required. In order to control the quality of the monolayer, its structure was studied on a microscopic and a macroscopic scale. The experimental methods were scanning tunneling microscopy (STM), wetting and electrochemical measurements. The chain length and the type of terminal group of the monolayer molecules were varied systematically.

    The microscopic structure of monolayers of alkanethiol (HS(CH 2 ) n -1 CH 3 with n = 3, 8, 12, 18, and 22) on Au(111) is the subject of chapter 3. This structure is investigated with atomically resolved STM and wetting measurements. The characteristic depressions in these monolayers as observed with STM are proven to be holes in the underlying top gold surface layer. These depressions are filled with thiol. The holes originate from an etching process of the gold during the adsorption of the thiol. A distinct correlation is found between the number of holes and the amount of gold in the thiol solution after adsorption, as measured with atomic absorption spectroscopy. The etching which generates these holes is believed to be related to the mobility of the gold-thiolate molecules during the adsorption process, prior to self-assembly.

    In chapter 4, the potential-dependent wetting of thiol-modified gold electrodes is for the first time presented. A Wilhelmy plate technique is used to determine the potential- dependent wetting of the modified electrodes. These measurements are carried out simultaneously with differential capacitance measurements and cyclovoltammetry. For alkanethiols with n >10, the monolayer is very stable in the potential range where only double layer charging occurs. The extreme hydrophobicity, the low dielectric constant (≈2 for n >10), and the low double layer current (about a factor of 100 less than for clean gold) are all indicative of the dielectric character of these monolayers.

    Chapter 5 reports on the influence of the alkanethiol chain length on the electrowetting effect of the self-assembled monolayer. It is found that the shorter the chain the stronger the wettability changes as a function of the potential. A simple representation of the electrical double layer as a dielectric thiol layer in series with a diffuse double layer in the electrolyte accounts well for the observed chain length effect. The effect of the salt concentration can be qualitatively understood with the model. It is concluded that the potential-dependent wetting finds it origin in the formation of an electrical double layer and that potential-induced conformational changes within the thiol layer are insignificant.

    Functionalizing the alkanethiols with a terminal group (HS(CH 2 ) n -1X , X = OH, CN, Cl and COOH) is found not to affect the stability of the monolayer, as follows from chapter 6. All thiols used are electroinactive except for the COOH group which can in part (5-10%) be reduced to the aldehyde compound. The difference in the capacitance of these thiol layers is determined by the different dielectric properties of the terminal group. The capacitance increases according to the sequence CH 3 3 Larger electrowetting effects are obtained by oxidation/reduction of a ferrocene- terminated alkanethiol monolayer, as described in chapter 7. Strong indications were found that the electrowettability is a result of specific anion binding upon oxidation of the ferrocene groups. This ion binding occurs to compensate the surface charge. The monolayers are not stable upon continuous oxidation/reduction of the ferrocene groups. The stability is strongly increased by mixing the ferrocenethiol with an alkanethiol of about the same chain length. The reversibility of the electrowetting is limited by contact angle hysteresis.

    The presence of a self-assembled thiol monolayer on a gold electrode strongly influences the morphology of galvanically deposited copper. This topic is discussed in chapter 8. On bare gold, copper is deposited as a rather homogeneous flat film, whereas on thiol-modified gold, independent of the type of terminal group, copper is deposited as hemispherical particles. The difference in morphology is ascribed to the difference between the surface energies of copper and the solid substrate. Generally, when a metal is deposited onto a solid material, flat films can only be obtained when the surface tension of the solid is high. Nucleation occurs on top of the thiol layer as long as the self-assembled monolayer is highly ordered. An overpotential is required to overcome the potential drop across the dielectric of the thiol layer. This potential drop increases with increasing thiol chain length and hence, the overpotential increases likewise. The influence of the terminal group of the thiol layer on galvanic copper deposition shows up most pronouncedly for the OH terminal group. About 100 times more particles are deposited on OH-terminated thiol compared to CH 3 -thiol. This is explained by a combination of a smaller potential drop across the thiol layer and a high chemical affinity of Cu atoms for the OH-group, thus decreasing surface diffusion.

    In the potential range between the equilibrium Nernst potential and the overpotential, no copper is deposited onto the monolayer. This makes the layers suitable as monolayer resists. In chapter 9 we focus on possible applications in this area. First, a thiol monolayer is treated by electron beam lithography to give very narrow patterns where the thiol has been removed. Subsequently, submicron metallized patterns can be produced by galvanic copper deposition in the openings. The smallest width of the copper patterns produced is about 75 run. The width is determined by the spot size of the electron beam. By optimizing the electron exposure, we expect that even finer patterns can be produced. Such fine metal structures may offer interesting applications in technologies such as ultra-high-density recording or disk mastering.

    This thesis concludes with an overview of this study and a comparison of our results with the current status of knowledge in the literature. In chapter 10, the information on the structure of the thiol monolayer as obtained with the various techniques is summarized. We conclude that methods like STM and metallization indicate the presence of small microscopic defects in the self-assembled monolayers. However, with electrochemical techniques like cyclovoltammetry and differential capacitance measurements it was shown that on a macroscopic scale the monolayers are ordered and densely packed. From the macroscopic point of view, the minor defects are shielded by the long chain alkane tails. Therefore, we have to conclude that the question whether a thiol monolayer can be considered as a model system depends on the particular type of goal one has in mind. In this thesis, we have demonstrated that thiol monolayers behaved as genuine model systems in the areas of electrowetting and monolayer lithography.

    Photosynthetic free energy transduction : modelling electrochemical events = [Fotosynthetische vrije energie omzetting : modelbeschrijvingen van elektrochemische verschijnselen
    Kooten, O. van - \ 1988
    Agricultural University. Promotor(en): W.J. Vredenberg. - S.l. : Van Kooten - 111
    fotosynthese - energie - overdracht - elektrochemie - photosynthesis - energy - transfer - electrochemistry

    This thesis is concerned with a particular part of the photosynthesis process. This part consists of the light-induced transmembrane electric potential gradient, the electrochemical pH gradient and the subsequent transformation of the energy contained in these gradients into chemical free energy of adenylates (ATP). This transformation of energy, originally in the form of an electromagnetic quantum, into a metabolic useful and stable chemical bond is called photosynthetic free energy transduction. After this process, which is sometimes inadequately referred to as the light reaction, the chemical energy is used, together with reduction equivalents from the electron transport chain, to bind protons and carbon molecules in order to form sugar molecules. In this last process, i.e. the Calvin cycle or the "dark reaction", free energy is not transduced, but shifted from one chemical bond to another. The reactions pertaining to the transduction of free energy are linked to the thylakoid membranes and are described in a general fashion in chapter 1.

    Three different approaches were used to study the free energy transduction. First the electrophysiological approach, i.e. measurements of the transmembrane electric potential with a microelectrode inserted in a single chloroplast. Second the spectrophotometric approach, in which a light-induced absorbance change of an ensemble of chloroplasts was studied with the aid of a spectrophotometer (P515). And third the modelling approach, from whence we have tried to encompass the host of experimental results in photosynthesis research in a rigorous mathematical formulation. The description of the methods used can be found in chapter 2.

    The basic model of photosynthetic free energy transduction, as it was originally developed to explain our electrophysiological results, is presented in chapter 3. It is based on the chemiosmotic hypothesis of membrane linked free energy transduction. Potential traces simulating the light-induced electric potential changes across the thylakoid membrane of an obligate shade plant like Peperomia metallica L. tend to reproduce experimental recordings measured with a microelectrode impaled in a chloroplast of that plant. A crucial role in this model is played by the formalism for the proton flux through the ATPase. The formalism developed by braber et al. (1984, 1987) for an oxidized ATPase was adapted to be applied in our model.

    Applications of the model and a comparison with earlier experiments are given in chapter 4. There it becomes evident that the model can be used for other techniques in photosynthesis research such as chlorophyll fluorescence induction curves. We compare simulations with microelectrode traces of the thylakoid membrane electric potential. Measurements by Remish et al. (1986a, b) of the combined pH gradient and the electric potential also confirm our model calculations.

    In chapter 5 we present electrophysiological (microelectrode) and spectrophotometric (P 515 ) results, which appear to challenge the premises on which the model presented in the previous chapters is based, namely Mitchell's hypothesis of chemiosmotic free energy transduction (Mitchell 1961). These results pertain to a deviation from a single exponential decay expected after a flash-induced rise in the electric potential. When measured with the microelectrode this is called a "new" response by us as opposed to the "classical" response. When measured with the P 515 technique it is called reaction II plus reaction I as opposed to reaction I alone. The P 515 response and the "new" response measured with the microelectrode show a striking similarity.

    In chapter 6 the phenomena underlying reaction II and the "new" response are explained. It appears that the Q-cycle, an explanation generally used for a particular component of reaction II (i.e. the relative slow rise), cannot be used to explain the P 515 measurements in view of the experiments and on theoretical grounds. We develop a model for reaction II based on stabilization of protons within the membrane matrix. This implies a dielectric separation of charges. Some charges reach the lumen and others stay within the membrane. The protons remaining within the membrane appear to leave the membrane via the membrane bound proton ATP synthase complex. This is incompatible with a basic assumption of the chemiosmotic hypothesis, i.e. all charges are transferred from the stroma to the lumen and both stroma and lumen are highly conductive and homogeneous phases. It appears that our model of reaction II is more in accord with an alternative hypothesis for membrane linked free energy transduction and usually referred to by the name of "localized" chemiosmosis (Williams 1961, Westerhoff et al. 1984). Although the "new" response measured by the microelectrode is similar to the P 515 response, it is explained by a different phenomenon. The structural effects of a microelectrode tip of 0.15 μm diameter penetrating a thylakoid membrane structure (granum) of 1.5 μm diameter is taken into consideration. This combined with the effect of a large concentration of "immobilized" buffer groups gives a completely different explanation for the "new" response.

    In chapter 7 we deliberate wether the "new" response and reaction II can be considered as an indication that the basic assumption of the chemiosmotic theory is violated. For the "new" response this is clearly not the case, since it is a consequence of the measuring method and its explanation can only lead to temporary inhomogeneities in the lumen. While for a true violation of the chemiosmotic theory the concept of a heterogeneous lumen must be sustained even under steady-state actinic illumination. For reaction II the answer is not so simple since an obvious connection exists between the membrane stabilized protons and the ATPase. However reaction II saturates after a short illumination period and stays saturated while the ATPase stays active. Thus it is not clear wether these protons play an active role during steady-state photophosphorylation. As was also shown by others it is likely that these protons play a role in the activation of the ATPase.

    Electrochemical analysis of metal complexes
    Jong, H.G. de - \ 1987
    Agricultural University. Promotor(en): J. Lyklema; H.P. van Leeuwen. - S.l. : De Jong - 76
    zware metalen - analytische methoden - elektrochemie - dubbelzouten - elektroanalytische analyse - heavy metals - analytical methods - electrochemistry - double salts - electroanalytical analysis

    The present study is concerned with the electroanalytical chemistry of complexes of metals with large ligands. The main purpose was to develop quantitative descriptions of the voltammetric current-potential relation of metal complex systems with different diffusion coefficients of the species involved and of the conductometric response of metal/polyelectrolyte systems at various metal-to-ligand ratios. A further goal was to illustrate the theoretical treatments with some experiments on model systems.
    In chapter 1 the general background of this study is discussed. After a brief review of some environmental aspects of heavy metals, the importance of reliable analytical techniques for their speciation is advanced. The particular potentialities of electroanalytical techniques in this field are outlined.
    In chapter 2 a rigorous theoretical treatment is given of the voltammetric limiting current for metal complex systems in which the diffusion coefficents of the species Involved are different. The equation for the limiting current is formulated in the Laplace domain and is rigorous with respect to the values of the association and dissociation rate constants as well as to the ratio of the diffusion coefficients of the species. It is valid for an excess of ligand and a stationary planar electrode. In some limiting cases analytical expressions in the time domain have been obtained. In the degenerate case of equal diffusion coefficients the limiting current can be described by a series expansion of confluent hypergeometric functions. Another limiting case is the situation where the diffusion coefficient of the ligand is infinitely small. Here, the use of a perturbation technique leads to a satisfactory approximation in the form of a series of confluent hypergeometric functions, for the case where the diffusion coefficient of the ligand is at least one order of magnitude smaller than that of the uncomplexed metal ion. Where analytical inversion of the Laplace transformed limiting current expression falls, transients are obtained by a numerical procedure. The results are identical with what may be obtained from the time-consuming fully numerical solution of the differential equations using the explicit finite difference algorithm.
    In chapter 3 the behaviour of the limiting current is investigated as a function of the electrolysis time, the ratio of the diffusion coefficients of the species and the association/dissociation rate constants. New qualifications to specify the voltammetric behaviour of metal complex system are introduced. Depending on the mean life-times of the species, the system can be static, semi-dynamic and dynamic. When the mean life-times are large compared to the electrolysis time, the system is static and the limiting current is proportional to the concentration of uncomplexed metal. In the opposite case where the life-times are small compared to the electrolysis time, the system is dynamic. A dynamic system can be further subdivided in labile and nonlabile. The lability concept unambiguously follows from considering the limiting cases in the theory. A new lability criterion is formulated, which is, compared to earlier tentative formulations, generalized to the metal complex system as a whole. In the non-labile regime the limiting current is found to follow exactly the classical reaction layer model, provided there is a large excess of complex over free metal. In order to facilitate practical use of the various results, some Cottrell plots and working curves are given.
    In chapter 4 a rigorous treatment of the complete current-potential relation of the metal complex systems from the previous chapters is given. The general relation is again formulated in the Laplace domain. Besides its limitation to reversible electrode processes, it has the same range of validity as the limiting current equation of chapter 2. The half-wave potential is obtained numerically from the general current-potential relation and its dependence on the electrolysis time, the ratio of the diffusion coefficients of the species and the association/dissociation rate constants is discussed. In the case of equal diffusion coefficients of the species, the current- potential relation can be analytically transformed from the Laplace- to the time domain. The result is given as a series expansion of confluent hypergeometric functions. In the dynamic regime the current-potential relation is described by an experfc type of function. An analytical expression for the half-wave potential is then obtained. In the case of a labile system and a large excess of complex this expression reduces to the well-known DeFord-Hume equation.
    In chapter 5 experimental data from earlier work on the voltammetry of heavy metal/polyelectrolyte systems are reconsidered. A procedure for solving a voltammetric speciation problem for a given set of values of the electrolysis time and the ratio of the diffusion coefficients is presented. Under the given experimental conditions the Zn/PMA system was found to be labile. Here the speciation is straightforwardly performed using the mean diffusion coefficient concept. The Cd/PMA data are substantially affected by induced adsorption of the metal ion. Until this is eliminated, interpretation is not possible. The Pb/PAA system seems to be dynamic and not labile. Here, the speciation is involved and performed by fitting the experimental data to the rigorous equations of the chapters 2 and 4 and as a result the order of magnitude of both the stability constant and the dissociation rate constant are obtained. Anticipating future presentations the correctness of the stability constant evaluated under the assumption of an excess of ligand, is estimated.
    In chapter 6 a procedure is described for the analysis of the conductivity of solutions of polyions in which both mono- and divalent counterions are present. The method is based on analysis of the relation between the overall conductivity of the system and the conductivity of the monovalent cations and assumes that the latter are electrostatically bound. The system is described in terms of the two state approach, implying that the counterions are considered to be either in some kind of bound state or are completely free. The potentialities of the proposed method are explored by studying solutions of alkali polyacrylates with and without added zinc nitrate at several alkali nitrate concentrations The results give a picture of the composition of the counterionic atmosphere around the polyion. Up to a certain Zn/polyacrylate ratio, the zinc ions were found to be bound quantitatively by the polyion. The composition of the counterionc atmosphere was largely independent of the alkali nitrate concentration if the latter was not in too large excess with respect to both the zinc ions and the charged monomers.

    Interfacial thermodynamics and electrochemistry of protein partitioning in two-phase systems
    Fraaije, J.G.E.M. - \ 1987
    Agricultural University. Promotor(en): J. Lyklema. - S.l. : Fraaije - 131
    capillairen - vloeistofmechanica - eiwitten - oppervlaktespanning - thermische energie - thermodynamica - elektrochemie - grenslaag - oppervlakteverschijnselen - tweefasesystemen - capillaries - fluid mechanics - proteins - surface tension - thermal energy - thermodynamics - electrochemistry - boundary layer - surface phenomena - two-phase systems

    The subject of this thesis is protein partition between an aqueous salt solution and a surface or an apolair liquid and the concomitant co-partition of small ions. The extent of co-partitioning determines the charge regulation in the protein partitioning process.

    Chapters 2 and 3 deal with phenomenological relations between the partition coefficient of the protein and the extent of the co- partition. The method of analysis is illustrated by some worked-out examples, using data taken either from literature or from chapter 5. The examples include proton titration curves, ion exchange chromatography, adsorption on colloidal particles and solubilization in reverse micelles. An important conclusion is that the partition process is subject to a rule, similar to the principle of Le Chatelier for chemical equilibria: if upon protein partitioning ions are expelled into the water phase, an increase of the ionic concentrations results in a decrease of the protein partition coefficient and conversely.

    A theory which allows for the prediction and molecular interpretation of the charge regulations is presented in chapter 4. The model describes the electrochemistry of a protein molecule through site binding of ions on a rigid surface. Although this is a considerably simplified picture of a real protein molecule, some aspects of the theory may be of general validity. One of them is the notion of the electrochemical adaptability of a charged colloidal particle, as measured by its intrinsic capacitance. In the case of a high intrinsic capacitance, a change in electrostatic interactions results in a large charge regulation whilst the surface potential remains almost constant. On the other hand, if the intrinsic capacitance is low, the particle resists externally imposed shifts in charge but does adapt its surface potential.

    Chapter 5 contains an experimental study towards understanding the mechanism of charge-regulation in protein adsorption. The system consists of crystals of the insoluble salt silver iodide as the adsorbent and the protein Bovine Serum Albumin as the adsorbate. By using a combined iodide and proton titration technique, the charges of the surface and the protein can be measured independently. We find that a negative surface induces a positive shift in the charge of the adsorbed protein. Opposed to intuitive expectation, the reverse is not always true: when the charge of the protein charge is maximally
    positive, adsorption renders the silver iodide surface less negative.

    The anomalous charge regulation is explained in terms of the intrinsic capacitance of the adsorbed protein. The maximally positive protein cannot adapt its charge, and so the silver iodide surface is forced to adjust its charge completely to that of the protein. As the contact layer between adsorbed protein and the silver iodide crystal is electroneutral under almost all circumstances, the silver iodide surface must be as negative as the protein is positive. Hence, if the charge of the surface before adsorption is more negative than this value, adsorption of the protein is accompanied by a desorption of negative charge.

    The experimental results are well understood in view of the developed phenomenological theory and model analysis. Two thermodynamic relations are succesfully verified, indicating the internal consistency of the various experiments. Application of the model gives two independent estimates of the size of the adsorbed protein. It is concluded that the protein does not substantially modify its native structure upon adsorption.

    Interfacial electrochemistry of colloidal ruthenium dioxide and catalysis of the photochemical generation of hydrogen from water
    Kleijn, J.M. - \ 1987
    Agricultural University. Promotor(en): J. Lyklema; H.P. van Leeuwen. - S.l. : Kleijn - 141
    ruthenium - colloïden - waterstof - elektrochemie - fotochemie - katalyse - ruthenium - colloids - hydrogen - electrochemistry - photochemistry - catalysis

    The formation of hydrogen from water using solar energy is a very attractive research topic, because of the potential use of hydrogen as an alternative, clean fuel. It has been shown by many workers in the field that photochemical hydrogen generation can be achieved in an aqueous system, containing a sensitizer (a light absorbing solute), an electron relay, and a dispersed catalyst. The electron relay transfers electrons from the light-excited sensitizer to the surface of the catalyst, where subsequent reduction of H +takes place. In an ideal photochemical system for solar energy conversion, water itself would ultimately provide the necessary electrons for hydrogen formation, under simultaneous oxygen evolution. However, complete ("cyclic") photodissociation of water involves a number of complications, like the recombination of intermediate photoproducts. To separately study the formation of hydrogen, these additional problems can be bypassed by adding an electron donor, which decomposes after having reduced the oxidized sensitizer. Such simplified systems are known as "sacrificial".

    The present thesis is concerned with the generation of hydrogen in such a sacrificial photochemical system. The main purpose has been to gain insight Into the processes that take place at the catalyst/solution interface. Because of its wide application in photochemical model systems for hydrogen production, methylviologen (MV 2+) was chosen as the electron relay. Via its reduced form MV +., electrons are transferred from the sensitizer to the catalyst. Colloidal ruthenium dioxide (RuO 2 ) was used as the catalyst compound. It has the advantage over the more commonly used Pt catalysts, that it does not catalyze the undesired, irreversible hydrogenation of MV 2+.

    The heterogeneous processes in a hydrogen photoproduction system cannot be investigated without taking into account the reactions in solution too. Therefore, ruthenium trisbipyridyl (Ru(bipy)32+) and EDTA were chosen as photosensitizer and sacrificial electron donor, respectively: most of the (light-induced) homogeneous reactions that take place in the Ru(bipy)32+/MV 2+/EDTA/colloidal catalyst system have been studied extensively by different groups of researchers. In our experiments, the standard reaction mixture (58 ml) for photogeneration of hydrogen contained 2 x 10 -4M Ru(bipy)32+, 5 x 10 -4M MV 2+, 0.02 M EDTA, and 0.05 M acetate buffer (pH 4.6).

    Colloidal RuO 2 was prepared by thermal decomposition of RuCl 3 at ca. 400 °C. The material obtained is crystalline and only slightly contaminated with residual Cl, which is mainly present at the surface of the particles. The BET surface area is 20-30 m 2/g. Dispersions of RuO 2 are colloid-chemically very unstable, even in the presence of polymers or surfactants. They manifest the same electric double layer characteristics as many other oxide dispersions. The point of zero charge (p.z.c.) in indifferent electrolyte (KNO 3 ) is positioned at pH 5.7-5.8.

    Experiments with RuO 2 film electrodes, prepared from the same colloidal material and sintered at 700 °C, revealed that the hydrogen evolution reaction is chemically reversible. Hydrogen evolution at moderate overpotentials does not modify the RuO 2 . In the presence of 0.05 M acetate buffer (pH 4.6), the mass transport limited current density for H +reduction is high since it is related to the buffer capacity and not to the actual proton activity. In the potential range studied, the hydrogen evolution reaction can be described by the Butler-Volmer equation, with a transfer coefficient αof about 0.33, and an exchange current density i o of ca. 0.09 mA/cm 2geometrical surface area. The true exchange current density is smaller by a factor depending on the surface roughness of the film electrodes.

    Adsorption, of MV 2+at the RuO 2 /solution interface is mainly a result of attractive coulombic interactions (above the p.z.c. of RuO 2 ), but it has been shown that there are also more specific interactions. However, the specific adsorption is weak and not noticeable at high concentrations of back-ground electrolyte and pH values below the p.z.c. of RuO 2 . No indications were found that MV 2+adsorbs at the catalyst surface under operational conditions of hydrogen evolution. Under these conditions, the sensitizer Ru(bipy)32+does not adsorb either. On the other hand, the electron donor EDTA strongly adsorbs on RuO 2 from a 0.05 M acetate buffer solution of pH 4.6. However, this seems not to affect the electron transfer between methylviologen and RuO 2 film electrodes, a process which takes place with a transfer coefficient αof ca. 0.35 and a standard heterogeneous rate constant k oof ca. 1.4 x 10 -5m/s (referred to the geometrical surface area).

    The colloidal RuO 2 turned out to be a good catalyst for photoproduction of hydrogen, in spite of the strong tendency of the particles to form aggregates. During the hydrogen evolution process, it does not loose its catalytic properties. It was confirmed that RuO 2 does not catalyze the hydrogenation of methylviologen. A disadvantage of RuO 2 is that it absorbs light throughout the entire visible region.

    Upon illumination of the reaction dispersion and after a certain induction time, hydrogen production takes place at a constant rate (steady state). After several hours, the production rate gradually decreases to zero. The maximum attainable amount of H 2 is determined by the initial amount of electron donor: each EDTA species can regenerate three oxidized sensitizer ions. However, in most experiments the total H 2 yield was less due to gradual destruction of methylviologen in the bulk solution.

    The steady state ratio [MV +.]/[MV 2+] appeared to be always low, even in the absence of catalyst. This must be the result of a yet unspecified reaction which reconverts MV +.into MV 2+. Probably, a photogenerated intermediate species is involved in this process.

    In all the experiments with the hydrogen photoproduction system, the incident light intensity was a rate-determining factor. The steady state rate of hydrogen production depends also, but to a lower extent, on the sensitizer concentration. It has been shown in a simple way that the first step in the hydrogen evolution process, i.e. the excitation of Ru(bipy)32+, is first order in the light intensity and less than first order in the sensitizer concentration.

    The hydrogen production rate increases with EDTA concentration up to a plateau above ca. 0.02 M. At the plateau, the oxidized sensitizer is regenerated efficiently, preventing back-reaction with MV + .As a function of methylviologen concentration, the production rate exhibits a maximum around 2 x 10 -3M.

    At low quantities of RuO 2 (< 10 mg), the available catalytic surface area is rate-limiting. At higher catalyst amounts, the production rate is fairly constant; it decreases slightly with increasing RuO 2 amount due to the absorption of light by the RuO 2 particles.

    For any amount of RuO 2 , the stirring rate affects the rate of hydrogen evolution. Mass transfer of H +to the catalyst surface is not rate-limiting, as is also confirmed by the insensitivity of the production rate to the buffer concentration. This implies that the mass transfer of MV + .to the catalyst surface is a rate-determining factor.

    Most of the abovementioned experimental results can be satisfactorily simulated using a quantitative model, in which the homogeneous reactions are described by steady state kinetic equations and the heterogeneous processes as electrode reactions. The catalytic properties of RuO 2 can be understood and predicted by considering the RuO 2 aggregates as microelectrodes. Probably, the electrical conductivity of RuO 2 -on the level of a metallic conductoris essential for its catalytic performance.

    Hydrogen evolution at the catalyst surface takes place near the equilibrium potential of the H +/H 2 couple. At these potentials, reconversion of MV 2+into MV + .at the catalyst surface is negligible. The rate of the heterogeneous processes is determined by the rate of mass transfer of MV + .to the surface and, to a lower degree, by the rate of interfacial electron transfer. The mass transfer coefficient of methylviologen, under the standard stirring conditions, appeared to be in the order of 10 -5m/s.

    Mass transfer of methylviologen would undoubtedly be favoured by a better dispersion of the catalyst, since aggregation of the RuO 2 particles makes the surface less accessible. If the same or higher hydrogen production rates could be reached with lower catalyst amounts, the disadvantage of light absorption by the RuO 2 particles would become less important. Therefore, it seems worth trying again to stabilize dispersions of RuO 2 , for example by covalently linking polymers to the oxide surface.

    The simulations further indicate that, if the total surface area of the RuO 2 particles is assumed to be catalytically active, the kinetic parameters i o and k oare only ca. 10 times lower than the corresponding values found for the RuO 2 film electrodes per unit geometrical surface area. This is surprising, because the roughness factor of these electrodes was estimated to be in the order of several hundreds. This point deserves further attention. Aspects that could be investigated, are the influence of heat treatments on the reductive catalytic properties of RuO 2 and the comparison with kinetic parameters for single crystal RuO 2 electrodes.

    The presented model for the hydrogen production system does not account for the maximum in hydrogen production rate as a function of methylviologen concentration. The differences between model predictions and experimental results point to a progressive inhibition of the heterogeneous processes with increasing MV 2+concentration. This aspect will be the subject of further study, including investigation of the dependency of the electron transfer rate constant on the bulk concentration of methylviologen.

    The overall quantum yield of the hydrogen production in our standard system is low; even with an excess of catalyst, it is less than 4 %. Since reconversion of MV 2+into MV + .at the catalyst surface does not take place (each MV + .species that reaches the surface is used for hydrogen production), the low efficiency of the system results from the homogeneous proceases. Reconversion of MV + .into MV 2+in solution is competitive with the production of hydrogen and makes the system less efficient. The quantum yield is also limited by the low efficiency of the quenching of the excited sensitizer by methylviologen. At pH 4.6, less than 25 % of the quenching acts results in charge separation (according to our numerical simulations ca. 16 %). Furthermore, the gradual destruction of methylviologen under illumination of the reaction mixture, makes this compound unsuitable for use in any practical device for photogeneration of hydrogen.

    Combination of information regarding the homogeneous and interfacial aspects of the hydrogen production system leads to a picture that is at least semiquantitatively, and in many aspects quantitatively consistent. Extentions of this approach could be useful for the rational design of catalytic systems for solar energy conversion.

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