THREE DIFFERENT SENSOR METHODS FOR METHANOL AND ETHANOL DETERMINATION.

Ethanol and methanol are two important alcohols used for different purposes. Consequently, also the analytical methods used to determine them are of considerable interest. Our team is therefore developing three different sensor-based methods for ethanol and methanol determination. Two of these consist of different enzyme electrodes and have been developed by immobilizing either alcohol oxidase or catalase in a kappa-Carrageenan gel layer overlapping an amperometric gaseous diffusion Clark type oxygen electrode. The variation of the oxygen concentration in the aqueous solution due to the enzymatic reactions was measured at a constant applied potential of – 650 mV. The responses of these biosensors toward standard solutions of methanol or ethanol were recorded, compared and discussed. In the case of the catalase electrode, the measurement was performed by adding hydrogen peroxide to a buffer solution which was diffused through the dialysis membrane towards the enzymatic layer where the reaction catalysed by the catalase enzyme took place: H2O2 1/2 O2 + H2O. Since this reaction led to the production of oxygen, the concentration of the latter in the measurement solution increased. This increase triggered an increase in the cathodic current measured which increased from the original value to a new value corresponding to a new stationary state. At this stage further additions were made (equal to 20 μL) of a standard solution of 0.008 mol L-1 of ethanol or methanol; after each addition a reaction of the following type occurred which was catalyzed by catalase: CH3CH2OH + H2O2 CH3CHO + 2 H2O. The second reaction removed part of the H2O2 substrate from the first reaction, which was slowed down; this slowdown was accompanied by a decrease in the level of oxygen produced in the solution during the first reaction; this decrease was evidenced by the decrease in the measured cathodic current which attained a new stationary state after each addition of alcohol solution. The current variation was read off after each alcohol addition [1]. With the biosensor operating with alcohol oxidase the operating procedure was much simpler as it consisted of directly making successive additions of the standard ethanol or methanol solution to a buffer solution in which the measurement was being performed. After each addition a reaction catalyzed by the alcohol oxidase enzyme of the following type took place: CH3CH2OH + O2 CH3CHO + H2O2. The reaction led to the oxygen present in the solution being consumed with a consequent decrease in the cathodic current measured until a new stationary state was reached. Also in this case, after each addition, the current variation was read off. All the experiments were carried out in a reaction cell thermostated at 23°C containing 15 mL of 0.05 mol L-1 phosphate buffer solution. The effect of pH on the response of the electrodes was investigated in detail and the best pH was found to be 7.5 for the catalase electrode and 8.0 for the alcohol oxidase electrode, respectively. The catalase biosensor displayed a much greater sensitivity to ethanol than to methanol, unlike the alcohol oxidase biosensor. It also displayed a stability and a life-time at least triple that of the alcohol oxidase biosensor as well as an LOD at least one decade lower. It also displayed a better repeatability and reproducibilty for ethanol and methanol solutions. The alcohol oxidase biosensor was instead found to be more sensitive to methanol than to ethanol but allowed the test to be carried out slightly faster than with the catalase device. In conclusion, our team is now investigating the feasibility of using a small catalytic ‘fuel cell’ originally constructed for the purpose of obtaining energy from methanol or ethanol but now adapted for analytical purposes [2,3]. The intention is to see whether this kind of device can also be used for ethanol and methanol determination. To this end water-alcohol solutions containing increasing percentages of ethanol or methanol are added to the cell, then following the potential increase that occurs at two electrodes of the cell and lastly reading off the maximum potential obtained with each addition. Although this part of the research is only at the preliminary stage it has already been possible to obtain calibration curves for both methanol and ethanol. A comparison of the linearity