Voltammetry

This Blog entry is intended to describe the basic processes of Voltammetry, and the application of this method to the analysis of enzymes. This information is summarised from the following sources: Elements of Molecular and Biomolecular Electrochemistry (Jean-Michel Saveant, pgs 298 to 316), Electrochemical Methods Fundamentals and Application (Bard and Faulkener, 2nd Edition). An understanding of chemistry equivalent to a typical undergraduate is assumed.

In analytical chemistry there are a wide array of analytical methods. These include mass spectrometry, spectroscpic methods, NMR analsis and so forth. In many cases such methods are innapropriate to the analysis being attempted. For instance if a compound is found in a solution that is optically dense i.e coloured spectroscopic analysis may not be useful. In this situation the solution would need to be seperated for the analysis to be performed. However if an analyte is redox active or is a an ion then it can be directly analysed using electrochemical methods. In particular voltammetry is useful in defining the characteristics of redox active molecules. This method is described below.

The basic instrument of electrochemical analysis is the electrochemical cell. A scematic of a simple cell is provided below.

(1)

Every electrochemical cell will contain 2 electrodes, a cathode and an anode. It will also contain an electrolyte solution, and a device to detect either current or voltage. In many cases, if the electrolyte for each electrode cannot mix the solutions will be connected by a salt bridge. In the case of electrochemical analysis one electrode will be designated the reference electrode and placed within a sealed area. This electrode is of a known type whose electrochemical characteristics are well established e.g. a hydrogen electrode. The other electrode is designated the sensor or working electrode and is placed in direct contact with the solution which is to be analysed. Voltammetry uses a standard electrochemical cell in it's processes. In addition this cell is connected to a power supply and an ampmeter. In this form of analysis a known potential (voltage) is applied to the sensor electrode and the resultant current is detected. This potential is then altered and the changes to current are recorded and analysed.

To describe what occurs let us suppose that the following reaction takes place.

O+e- --> R

That is the oxidised form of the redox active molecule being analysed is reduced at the electrode surface. Via mass transport the analyte moves towards the elctrode. When in contact with it the analyte is reduced and then moves away via mass transport. The current detected is the rate of electron tansport. This rate will depend on the temperature, the rate of diffusion of particles in the solution etc and can be represented mathematically by the Cottrell equation presented here. (2)

Where I=the current detected in Amperes.

n=number of electrons transferred

F=Faraday Constant, 96,500 C/mol

A=Electrode surface area, cm3

cjo=Initial concentration of analyte, j, in oxidised form, mol/cm3

Dj= Diffusion coefficient for analyte j, cm2/s

t= Time, s

The oxidation of analyte at the electrode, whereby electrons are transferred from the analyte into the elctrode is also possible. Which occurs will depend on the electrode potential. Should the potential applied be more negative than the reduction potential of the analyte it will be reduced. Should it be more positive than the reduction potential it will be oxidised. Should a negative potential be applied to the already reduced form then no current will be detected, the reverse being true about the oxidation of the reduced form. Finally the reduction of the analyte at the electrode will result in a negative current, the oxidation of the analyte will yield a posotive current. Whilst many methods of varying the potential exist the one most important to this study is cyclic voltammetry. In this method the voltage is varied in a linear fashion from more positive than the reduction potential to significantly more negative and back again. The resultant current is then recorded and analysed. The potentials are chosen to ensure both complete oxidation and reduction during the experiment to allow for a more detailed analysis. Typically the data can be presented graphically, such as below.

(3)

As you can see the potential is shown along the x-axis and the current along the y-axis. The shape shown here is distinctive of a single electron transfer. A wealth of information can be gained from such a voltammagram including the reduction potential, electron stoichiometry and so on. This will be demonstrated in a later post as a voltammogram is interpreted.

To conclude a brief mention shall be given to voltammetry as conducted upon enzymes. It is known that enzymes can achieve a direct electon transfer with the electrode surface without excessive difficulty. A more important factor is the problem of diffusion and mass transport for such massive molecules. Such problems can be overcom by adhering the enzyme to a specific membrane which is present at the electrode surface. The conditions for this are clearly explained in "insert source". In summary though, if a membrane is selected which is very thin (typically monolayer) and allows free access of the active site of the enzyme to both the solution and the electrode surface an electrochemical analysis can be achieved. What this means is that the structure within the enzyme appropriate to electron transfer (such as the heme group of a cytochrome protein) must be placed to allow a free flow of electons between the group and the electrode. Thus the membrane must not restrict this movement which can be achieved by direct access or conformational changes within the enzyme. A further advantage of this method (in addition to removing mass transport issues) is that a relatively small amount of enzyme is required for the analysis. This allows for meaningful analysis of enzymes which are available only in small quantities or at high prices.

Notes:

1) Image taken from http://www.doitpoms.ac.uk/tlplib/pourbaix/images/electrode_potential.gif

2) Equation image taken from http://en.wikipedia.org/wiki/Cottrell_equation. Checked against various sources.

3) Image taken from http://en.wikipedia.org/wiki/File:Cyclovoltammogram.jpg.