4.

CALIBRATION

The electrochemical reactions generate a minute ‘residual current’ even in the

absence of oxygen and if accurate calibration is required the discrepancy between

zero oxygen and the electrical zero should be identified by passing nitrogen over the

electrode. Alternatively a small drop of dithionite solution may be placed on the

cathode. This consumes O2 according to the equation

and, since the reaction goes virtually to completion, this procedure is equivalent to

flushing with oxygen-free gas. Unfortunately, dithionite is not pleasant to use

(CAUTION, DITHIONITE IS CORROSIVE) and it can also cause damage to, or

poisoning of, the membrane. For these reasons it is best avoided. If there is no

alternative, it should be removed from the membrane as quickly as possible by using

an aspirator with a soft tip (to prevent mechanical damage).

In this way, the true zero is established and, if preferred, this can be “backed-off” to

coincide with electrical zero. Once the dithionite solution is removed (or N2 flushing

is stopped), and the cathode is exposed to air, the signal generated by 21% oxygen

(the oxygen content of air) can be determined. In a closed system the amount of O2 is

governed by the partial pressure of O2 and the volume of the chamber. Thus 1 ml

(1000 μl) of air will contain 210 μl of oxygen. Similarly, if 1 ml of the space, in a 5 ml

chamber, is occupied by a leaf-disc the increase in signal which follows the

introduction of a given volume of air into the chamber will be correspondingly

greater. In fact, the difference between the two signals can be used to determine the

volume of a leaf-disc with some accuracy (4b and Fig. 4.2). In general, however,

although allowances can be made for differences in atmospheric pressure etc., the

instrument was designed for simplicity rather than absolute measurement of oxygen.

Although it has proved to be much more versatile than was originally imagined it was

originally intended for the measurement of the oxygen evolved when a relatively

large piece of leaf was illuminated in a relatively small chamber. For most purposes,

therefore, the excursions following the introduction (or “flushing

removal) of small volumes of air into the chamber (using a gas-tight syringe) can be
taken as the basis of calibration and all that remains to be done is some simple

arithmetic (see Fig. 4b). Experiment 1 incorporates several aspects of the calibration

procedure but this can be shortened (4e) or extended according to need and the degree

of accuracy sought.

4(a)Experiment l. Does it work?

Using a 1 ml gas-tight syringe, introduce successive 200 μl aliquots of air into the

leaf-disc chamber through one of the two gas-vents. (The other should be kept closed

during this procedure). As each volume of air is pushed into the chamber the reading

on the pen-recorder should rise quickly to a new level and stay there (Fig. 4.1). You

will see that the initial rise is fast but that it decreases as it approaches its final level.

This is because the rate of diffusion of oxygen to the detector will become limiting as

the difference in concentration between the atmosphere in the chamber and that at the

electrode surface approaches equilibrium. If equilibrium is not approached within

about one minute (i.e. if the response is sluggish) the electrode may require cleaning.

If the signal rises rapidly but then falls equally quickly, the chamber is leaking and

you should check to see that you have remembered to close the other gas-tap, that the

electrode disc is pressed securely against its O-ring, that the O-ring which seals the

top of the chamber to the bottom is in place and that the clips which hold the two parts

of the chamber together are properly closed. If the response seems too slow for the

experiments that you have in mind, it can be speeded by using a thinner membrane, by

cutting a hole in the spacer immediately above the cathode or by dispensing with the

spacer altogether. If no spacer is used, departure from linearity may sometimes result

(see below).

Figure 4.1. Excursions produced by introducing and removing aliquots of air.

A 1 ml, gas-tight, syringe, with its plunger fully withdrawn (to the 1 ml position) was

attached to an open tap on the leaf chamber and the other tap closed. The figure

illustrates the responses which were observed as the plunger was then pressed

inwards in 200 μl stages and finally withdrawn again in a similar fashion. Note that

each successive addition produced a larger signal (for explanation see text