Thin Film Cyclic Voltammetry

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Thin Film Cyclic Voltammetry

description

Thin Film Cyclic Voltammetry. Equipment for film voltammetry. potentiostat. electrode material. insulator. reference. Electroactive film. N 2 inlet. counter. working electrode. E-t waveform. Cyclic voltammetry. E, V. Electrochemical cell. time. - PowerPoint PPT Presentation

Transcript of Thin Film Cyclic Voltammetry

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Thin Film Cyclic Voltammetry

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E, V

time

E-t waveform

potentiostat

Electrochemical cell

counter

working electrode

N2

inlet

Electroactive film

reference

insulator electrodematerial

Equipment for film voltammetry

Cyclic voltammetry

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Ideal, reversible thin layer cyclic voltammogram

Example cobalt complex: LCoIII + e- LCoII

Q = nFAGT GT = total surface concentration of electroactive speciesA = electrode area, F = Faraday’s constant

reversible peak current Ip increases linearly as scan rate () is increased;And DEp = 0. Rate constants can be obtained by increasing to drive the CV into a kinetically limited situation where DEp > 0. Q = area under reduction curve

Ep

Ip

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Ferrocene SAM Electroactive polymer

Many types of electroactive films

Protein SAM

SAM = self assembled monolayer

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Ideal, reversible thin layer cyclic voltammogram

Example cobalt complex: LCoIII + e- LCoII

Q = nFAGT GT = total surface concentration of electroactive speciesA = electrode area, F = Faraday’s constant

reversible peak current Ip increases linearly as scan rate () is increased;And DEp = 0. Rate constants can be obtained by increasing to drive the CV into a kinetically limited situation where DEp > 0. Q = area under reduction curve

Ep

Ip

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Real CVs, includeCharging currentAnd some non-ideality

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electrode

Protein (monolayer)

Apply voltage Measure current

Thin Film Electrochemistry of Proteins

Information obtained:1. Redox potentials, free energies, re-organization energies2. Redox mechanism: protonation/deprotonation and chemical reaction steps3. Kinetics and thermodynamics of catalytic reactions4. Biosensors

Electrochemistry of proteins in solution• electrode fouling, proteins denature• large size means small D, tiny signals• need lots of protein

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Electrode

enzyme

A lipid-protein film

One way to make a stable protein film

• Many other types of films possible - polyions,Adsorbed, crosslinked, etc.

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OxidationOf FeII

ReductionOf FeIII

Reversible Peaks for

Direct electronTransfer;

Peak shapes, sizes, and Ep

reveal details of redox chemistry

Nearly idealReversible ET

Forward peak

Reverse peak

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LbL

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Kinetically limited CV at 0.1 V s-1 for 40 nm myoglobin (Mb)-polyion film on a PG electrode in pH 5.5 buffer at 35 oC. Example where rate constants can be obtained by increasing to drive the CV into a kinetically limited situation; DEp >> 0. Mb is another iron heme protein, peaks are for redox reactions of iron.Value of ks (s-1) cas be obtained by fitting data to theoretical curves of DEp vs. log scan rate or by fitting with best fit digital simulations of the CVs.

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Cytochrome P450 Enzymes

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Prof. John Schenkman, Pharmacology,Cell Biology, Uconn Health Center

Human Metabolic Enzymes:

CytP450s in LbL polyion films: • ET reduction rates from CV depend on spin state of cyt P450 iron heme (low spin fastest); conformational equilibria• rates of oxidation by peroxide depend on spin state (high spin fastest) and secondary structure

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Thin Film voltammetry of human cyt P450s

LbL films of cyt P450s and polyions on pyrolytic graphite electrodes. Polyions are purple strands and proteins are green/red ribbons . Thickness 10-25 nm

Sadagopan Krishnan, Amila Abeykoon, John B. Schenkman, and James F. Rusling, Control of Electrochemical and Ferryloxy Formation Kinetics of Cyt P450s in Polyion Films by Heme Iron Spin State and Secondary Structure, J. Am. Chem. Soc. 2009, 131, 16215–16224.

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Spectral characterization of cyt P450 films

UV-vis spectra of cyt P450 films on aminosilane-functionalized fused silica slides: (A) CO difference spectrum confirming native protein in PEI(/PSS/cyt P450 1A2)6 film after reducing to the ferrous form and purging the pH 7 buffer with CO; (B) ferric high spin form of enzyme in PEI(/PSS/cyt P450 1A2)6; and (C) ferric low spin form of enzyme in PSS(/PEI/cyt P450cam)6 film.

PFeII-COPFeIII PFeIII

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Cyclic Voltammetry and rate constant (ks) estimates Assuming simple electron transfer model

P450 2E1 P450 cam

Background subtracted cyclic voltammograms of LbL films on PG electrodes in anaerobic 50 buffer + 0.1 M NaCl, pH 7.0

Rate const. estimation for cyt P450/polyion films experimental () peak separation (Ep) corrected for scan rate independent non-kinetic contribution. Lines for Butler-Volmer theory for the rate constant (ks) shown and a= 0.5.

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Lines were from digital simulation using

The simple reversible theory did not fit peak potential vs. scan rate data, so complex model

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Conclusions for cyt P450 ET from thinFilm voltammetry:

• low spin cyt P450cam, ks = 95 s-1

mixed spin cyt P450 1E2, ks = 18 s-1 (80% high spin) high spin cyt P450 1A2, ks = 2.3 s-1

• ks for the reduction step correlates with spin state of the iron heme in the cyt P450, as found for solution reductions

• rates of oxidation by peroxide depend on spin state (high spin fastest) also

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Divided cell – keep products apart

Undivided cell – sacrificial anode can be usede.g. Cu Cu2+ + 2e

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Divided Electrolysis Cell for synthetic use

Large working electrode + refCounter electrode

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