Chapter 1: General Introduction NaC
2.1/ Introduction
2.2.2/ The ECD system
2.2.2.1/ECD, Theory
Once the solutes have crossed the column, they enter the electrochemical cell where they are detected. The cell used in this study consists of two glassy carbon electrodes in series. A negative potential is applied at the first one so as to condition the mobile phase containing the solutes. A positive potential is applied to the second one where the reaction of detection takes place (an oxidation).
The transfer of electrons (Figure 2.3) at the electrode during the oxidation gives rise to a current measured by the detector and expressed as a peak on a chromatogram by the integrator (Figure 1.7). According to Equation 2.1, the current obtained is proportional to the difference between the rate of the oxidation and reduction reactions at the electrode (Brett & Brett, 1993).
Electrode surface Electron transfer Adsorption Ox kc Red kd. Ox < = C > O’' Diffusion kd . Red < > Diffusion
Figure 2.3 Scheme o f electron transfer at an electrode.
Adapted from Brett & Brett, (1993).
Red* and Ox« are, respectively, the concentrations at infinite distance (in the bulk solution) of the reduced and oxidised form of the substance. Red. and Ox* are, respectively, the concentrations at the electrode surface of the reduced and oxidised form of the substance.
Equation 2.1 I = nFA(ka[RedJ* - kc[Ox] ♦)
F: Faraday constant A: Area of the electrode
n: number of electrons exchanged during the reaction
By changing the potential E, we change ka and kc, and I varies accordingly. However, the transport of electroactive species close to the electrode constitutes a limitation and the current I cannot increase above a maximum value called the diffusion- limited current (see Figure 2.4). This current is proportional to the concentration o f the solute. Hence, if one wants to be able to evaluate the concentration of a solute in a sample, it is necessary to work at a potential, E, such that I = lL,a- In such conditions, the reaction taking place is the oxidation of the reduced species.
Chapter 2: Methods
Oxidation
L.a
I L.c
Reduction
Figure 2.4 Voltammogram fo r a reversible system where the solution contains the oxidised and the reduced species.
+ (RT/nF) In (k d. Red / k d, ox)
with: E eq = E + (RT/nF) In ([Ox]«, / [Red]*) Nemst Equation The following equation is valid for any uniformly accessible electrode: Equation 2.2 E = E .//+ (RT/nF) In ((I l.c - I)/(I - I La))
2.2.2.2/ Choice of the potential of oxidation
According to previous studies done by colleagues, the conditioning electrode located in the electrochemical cell was set at -280 mV. In order to determine the optimal potential for the second electrode, a current/potential calibration curve for noradrenaline was constructed.
The HPLC system was set up with the characteristics described in section 2.2.1.3 and the protocol was as follows: a noradrenaline sample from a stock solution o f a concentration of 100 ffnol/50 pi was injected three times into the system which was set at a potential ranging between -50 and +350 mV. The potential was then changed and, after the system had settled down, the operation was repeated. The stock solution of noradrenaline was made in perchloric acid O.IM and kept in ice to minimise the decay of noradrenaline.
The voltammogram obtained (Figure 2.5) by oxidation of noradrenaline (Figure 2.6) showed a plateau from 350mV down to about 120mV. At this potential, the height of the peak on the chromatogram began to decrease severely in a linear relationship with the potential E. >
I
• # II m « 0.8 - c I 0.6 - Ü c g 0.4 - 0.2 - 0.0 -100 0 100 200 300 400 E(mV)Figure 2.5 Voltammogram obtained by oxidation o f noradrenaline at potentials ranging between —50 and +350 mV.
OH
OH
+
OH
Figure 2.6 Chemical equation o f oxidation o f noradrenaline.
It was decided to set the measuring electrode at 180 mV for the future experiments. This potential should be high enough to ensure that the diffusion-limited current is reached (see section 2.2.2.1) and low enough to prevent the oxidation of too
many impurities whose detection signals could interfere with those of the substances measured.
Chapter 2: Methods
For comparison, a voltammogram fo r 5-HT was also constructed. The protocol was identical to the one used to construct the graph fo r noradrenaline and the HPLC system was set at the conditons described in section 2.2.1.2, with a concentration o f DSA o f 0.15 mM. The concentration o f the stock solution o f 5-HT was 400fmol/50 pd.
The voltammogram obtained fo r 5-HT (Figure 2.7) presented the same profile as that for noradrenaline: a plateau from 350 mV down to about 90 mV, followed by a steep decline in a linear relationship with the potential E. This showed that if 5-HT alone was to be detected, the potential at the second electrode o f the electrochemical cell could be set at a lower value than that fo r measuring noradrenaline.
1.4 - E 1 .2 -
1
II 1 .0 - LU ♦♦♦♦ «3 0 . 8 -1
Ü 0 . 6 -1
0.4 - ♦♦ 3 Ü 0 . 2 - ♦♦ 0 . 0 - 1 1 -100 ♦ ♦ ♦ ♦ 100 200 300 400 E(mV)Figure 2.7 Voltammogram obtained by oxidation o f 5-HT at potentials ranging between -5 0 and +300 mV.
2.2.2.3Z Characteristics o f the ECD system
In the present study, the ECD system consisted in:
a guard cell ESA model 5020 set at +350 mV
an electrochemical cell: model ESA 5014 a consisting of two electrodes in series:
• a conditioning electrode set at -280 mV • a measuring electrode set at 180 mV a Coulochem detector: ESA 5100 A
an integrator: Spectra-Physics Chromjet integrator or TurboChrom package
The cell used in this study is a coulometric sensor: i.e. 100% of the analyte reaching the cell undergoes electrolysis (Figure 2.8). Therefore, according to Faraday’s law, the peak area on the chromatogram is directly related to the quantity of sample injected. However, the quantities measured during this study were sometimes so low that the peak could not be evaluated by the integrator. For this reason, it was decided that the height of the peak would be used to reflect the concentration of dialysates and that this parameter would always be measured manually.