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 PresentationTRANSCRIPT
Thin Film Cyclic Voltammetry
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
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
Ferrocene SAM Electroactive polymer
Many types of electroactive films
Protein SAM
SAM = self assembled monolayer
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
Real CVs, includeCharging currentAnd some non-ideality
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
Electrode
enzyme
A lipid-protein film
One way to make a stable protein film
• Many other types of films possible - polyions,Adsorbed, crosslinked, etc.
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
LbL
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.
Cytochrome P450 Enzymes
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
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.
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
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.
Lines were from digital simulation using
The simple reversible theory did not fit peak potential vs. scan rate data, so complex model
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
Divided cell – keep products apart
Undivided cell – sacrificial anode can be usede.g. Cu Cu2+ + 2e
Divided Electrolysis Cell for synthetic use
Large working electrode + refCounter electrode