Winkler Method (by Walter)

The oxygen is a fundamental element for the live in earth. The his amount in air, and in oceanic superficial layer, is 21% in constant balance in gas exchange air-sea.
The Winkler method is a test to determinate the concentration of dissolved oxygen in water samples: the quantity of dissolved oxygen is one of the measures of biological activities in seawater or freshwater. It’s necessary also, for example, to study water masses in the ocean or to measure the redox potential in water column. This test was originally developed by Lajos Winkler, an Hungarian analytical chemist, in 1888, modifying a preceding test. Winkler discovered a safer and more precise method of dissolved oxygen analysis thanks to an iodometric titration.
Carpenter in 1965 modified the Winkler method for analysis of dissolved oxygen, because he found some particularities about accuracy and errors, like air oxidation of iodide and volatilization of iodine, oxygen contributed by the reagent solution, iodate contamination of the iodide solutions, consumption or production of iodine by reagent contaminants, difference between titration end point and the equivalence point.

Principles of analysis
The production and respiration rates of microbial community will be calculate by variation in amount of dissolved oxygen in a defined time and comprehensive of all organisms activities in samples (autotrophs and heterotrophs).
The samples taken from mesocosms, will be fixed with a sequence of MnCl2 and NaI, resulting in a brown precipitate, and will be preserve a part in shadow and a part in light at same sea temperature on 24 h. The difference on amount of dissolved oxygen between T24 and T0 in shadow gives an estimate of planktonic respiration, a biological process of all organisms (in our case planktonic) where it obtains energy from oxidation of reduced organic compounds, realising CO2. Instead, the difference on amount of dissolved oxygen between T24 and T0 in light gives an estimate of planktonic production, a biological process of organisms that produce, by photosynthesis, organic molecules from carbon inorganic, by a reduction reaction, realising O2 (the photosynthetic organisms, primary producers, are at lower trophic level, supporting the total ocean and terrestrial life forming biomass).
After the incubation, it will be necessary proceed with a iodometric titration, because one mole of O2 reacts with four moles of thiosulfate and, by calculation of amount of thiosulphate, it’s possible calculate how many dissolved oxygen is in samples. Here I will try to explain shortly how.
Prior to start the analysis, it’s necessary standardize the titrator with a standard solution of KIO3 (potassium iodate), to minimize the error of the machine and to know the right concentration of the titrant solution. Moreover the sample is acidified with H2SO4 (sulfuric acid) to dissolve the hydroxides precipitated (MnCl2 + NaI) , liberating elementary iodine (I2) that reacts with surplus iodide ions, forming a complex (I3-) that is titrated with sodium thiosulphate. The total reaction is this following:
2S2O32- ↔ I2 ↔ ½ O2

Phases of the method

Calculation and expression of results
With the iodometric titration, the titrator by the Tiamo software calculates the amount (equivalent volume) of thiosulphate that is necessary in titration at equivalent point of our samples. The calculation will be done by Excel, in which considering the real volume of bottles (approximately 50-60 ml) and putting the equivalent volume, by a formula it’s possible know the right oxygen amount (μmol/L). Furthermore, calculating the mean and standard deviation, crossing the fingers, we can do some considerations about our results.

Winkler Team

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