LIMITATIONS OF DYES 27 



LIMITATIONS IN THE USE OF OXIDATION-REDUCTION POTENTIAL INDICATORS 



With the help of data of the type presented in the foregoing tables, the choice 

 of dyes of suitable Ei^ range is possible. It is now necessary to apply other criteria. 

 The dyes chosen should have a distinctive colour readily observable in the presence 

 of biological fluids, and should not behave, like some of the simpler indophenols, 

 as pH indicators (Fildes, 1929). Such dyes, although of an intense blue colour in 

 alkaline solution, may in the biological range of pH near the neutral point have a 

 dull reddish brown tint, which is barely distinguishable from that of tissue fluids or 

 culture media. Such dyes are, of course, almost useless to bacteriologists and 

 biologists generally. 



When, however, dyes have been selected of suitable E^ range and which have 

 a distinct colour at the pH of the experimental system, it is necessary to consider 

 further possible limitations and fallacies in interpreting the result obtained. 



Capacity and Poising Effects 



Many biological systems, although having a clear-cut and definite level of 

 potential, may be of small effective oxidation-reduction capacity, and not be well 

 poised. (This is equivalent to a solution being not well-buffered in pH determina- 

 tions.) In these cases, when the oxidised form of th^ dye is added to the system, 

 the dye is capable of oxidising the system entirely without itself being reduced 

 appreciably so that the level of potential of the system approximates to that of the 

 oxidised form of the dye. This, really, follows from consideration of the electrode 

 potential equation. Let us consider the electrode equations of the biological 

 system (B) and of the dye (D), and let them both be of the same form, at 30°C., 

 and at the same pH. 



The system Eh = E^ -f 0-03 log [^ 



and the dye Eh = E'J + 0-03 log [^^ 



The level of potential of each is dependent solely on the ratio of the concentrations 

 of the oxidised and reduced form. That is to say, that at any given degree of 

 oxidation, solutions of the dye will have the same E h, no matter whether the dye 

 solution is of 0-001 per cent., 1 per cent, or 10 per cent, concentration, and the same 

 apphes to the system. But if now the two solutions are mixed, their actual total 

 concentrations become of importance. Should the dye contain 100 gm. equivalent 

 per litre and the biological system only 1 gm. equivalent per litre (of the oxidation- 

 reduction system responsible for the potential) the dye can oxidise the whole of the 

 biological system and still remain 99 per cent, oxidised, thus proving useless as an 

 indicator of the condition of the system. 



The relative capacities, or poising effects, of the experimental system and of 

 the oxidation-reduction potential indicator must therefore be taken into account 

 in the interpretation of results. 



Time Factor 



A considerable time may elapse before the biological system comes into 

 equilibrium with the indicator. This is due in part to the method of establishment 



