The reaction of Mn(II) with water-dissolved oxygen, to a higher manganese hydroxide in an alkaline medium, as with the longstanding classic Winkler method, is the first step in the method described here. The assumption for faultless results by the conventional and modified Winkler method is clean water, which contains no organic substances by Mn(III) or Mn(IV). In many cases, however, eg. in river and lake-water tests, it can be seen with the naked eye that after some time the originally brown-coloured precipitate of manganese hydroxide becomes more and more colourless. Oxygen content was analysed in the water samples and evaluated by raising the amount of the leuko-base and giving the corresponding dilution of the colouring matter solution formed still higher oxygen contents can be measured

Altmann, H. J.

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Title: Determination of dissolved oxygen in water with Leukoberbelin-Blue I. 
A quick Winkler method. 
Author(s) H. J. Altmann 
Reference: Z. analyt. Chem. 262, 97-99 
Original language: German 
Date of publication of original: 1972 
Translators Lorna Heller 
Date of publication of translation: 1974 
Number of pages of translation: 5 
FBA Translations are to be considered as "provisional". As a rule 
they have not been prepared by expert translators, nor have they 
been edited by the original authors. 
They are available from The Librarian, 
Freshwater Biological Association, 
The Ferry House, 
Far Sawrey, 
AMBLESIDE, 
Westmorland, 
England, 
at the cu r r en t r a t e f o r xerox copying. 
Freshwater B i o l o g i c a l Assoc ia t ion 
FBA T r a n s l a t i o n (New S e r i e s ) No. 68 
FBA Translation (New Series) No. 68 
ALTMANN H.J. (1972). Determination of dissolved oxygen in water with 
Leukoberbelin-Blue I. A quick Winkler method. Z. analyt. Chem. 262, 97.99. 
Translated by Lorna Heller 
1. Introduction 
The reaction of Mn(II) with water-dissolved oxygen, to a higher 
manganese hydroxide in an alkaline medium, as with the longstanding classic 
Winkler method, is the first step in the method described here. The 
assumption for faultless results by the conventional and modified Winkler 
method is clean water, which contains no organic substances by Mn(III) or 
Mn(IV). In many cases, however, eg. in river and lake-water tests, it can 
be seen with the naked eye that after some time the originally brown-coloured 
precipitate of manganese hydroxide becomes more and more colourless. The 
time which elapses before the precipitate of manganese hydroxide has settled 
is sufficient to allow a part of the freshly precipitated and very active 
higher manganese hydroxide to react with the organic content of the water. 
The consequences of this are false measurements. The reaction O2 + Mn(OH)2 --> 
higher manganese hydroxide comes to an end quantitatively in a few seconds and 
with it ends the basic reaction of the oxygen determination. Any continuing 
contact of the higher manganese hydroxide with the organic material in the 
water is detrimental and is only necessary so far because the manganese 
hydroxide must settle for the continuation of the further steps of the method. 
2. Theoretical Part 
Leuko-bases of triphenylmethane colouring substances are oxidised in 
the space of a few seconds in an acidic medium by Mn(III) or Mn(IV) to 
coloured cations. The extinction of solutions of colouring matter so 
obtained is consequently also a measure for the determination of the oxygen 
content. The leuko-bases of indigo-carmine and the o-toluidine have hitherto 
been used in similar methods. These reactants are, however, either unstable 
themselves or do not form stable colouring substances. This is also true of 
leuko-malachite green used for the determination of manganese, as described in 
the literature, which besides is not soluble in water (1, 2, 4). 
Further triarylmethane-derivatives have been examined as reagents for 
the determination of Mn(VII) (3). 
We have succeeded, through the synthesis of the leuko-base of a 
triphenylmethane substance, in removing these troublesome factors. We have 
obtained a new reagent, which is suitable for the determination of the oxygen 
content of a water specimen. Mn(0H)2, reacts in a well known way to form 
higher manganese hydroxides, characterized here by MnO2. The leuko-base 
(FH) of the colouring substance is oxidised quantitatively to the colouring 
substance (F+) in this way. 
The reagent Leukoberbelin-Blue I (FH) used by us, is 2-(bis-
(4-dimethylaminophenyl) methyl) benzene sulphonic acid. Thus the reaction 
is comprised as follows: 
1 
3. Synthesis of Leukoberbelin-Blue I 
25g (25ml, 0.21M) freshly distilled N-dimethylaniline and 21g (ca. 
0.1M) 2-formylbenzol sulphonic acid (sodium salt) are reflux-condensed with 
30ml 2N H2SO4 for 24 yours. Then they are cooled, neutralised with 4N NaOH, 
and the excess N-dimethylaniline is driven off with steam. After cooling, 
the crystal sludge is rigorously vacuum filtered and washed with a few 
milliliters of water. The still damp substance is recrystalised from 
methanol. Particularly pure preparations are obtained if the acetone 
solution of the leuko-base is filtered through a silica gel column. Should, 
after some time (several weeks), a surface blue-colouring occur on the crystals, 
the substance can be repurified by filtration through silica gel. 
Yield: 29.4 g (71.6%) 
Formula: C 2 3 H 2 6 O 3 S Mwt. 410,54 
λ max of the colouring substance 623 nm 
Furthermore, the N-dimethylaniline and N-phenylmorpholin are converted with 
formylbenzol-2,4-disulphonic acid (sodium salt) by the above method. Both 
products of the synthesis are also useful as reagents, but more difficult to 
produce purely. 
4. Behaviour of Leukoberbelin-Blue I and its associated colouring substances 
The pure substance of Leukoberbelin-Blue I, when stored in the dark, 
can be kept for several weeks, during which time it slowly forms the 
associated colouring matter. The aqueous solution of the leuko-base, with 
a few drops of NH4OH solution added, is stable for over a year. The 
extinction of the colouring substance Berbelin-Blue I formed in the reaction 
is not altered in the acetate buffer. The analysis can consequently take 
Available through: Mecron GmbH, 1 Berlin 4l, Haupstr. 76. 
2 
Fig 1. Reaction vessel for the oxygen determination. 
place of the sampling, while the measuring of the colouring matter formed 
can still be undertaken days later in another place. The colouring matter 
has a molar extinction coefficient of ε = 110,000. 
5. Apparatus and chemicals 
For the determination of the dissolved oxygen, several syringes 
(polyethylene) with 7 cm long, sharp cannules and the glass vessel (ca 100 ml) 
shown in fig. 1 are necessary. In one ground opening a suitable stopper 
(brass) is fitted, sealed with silicone grease, which has in the bottom a 
packing of silicone rubber - (packing rubber for gas chromatography, made by 
Perkin and Elmer). This packing is pierced from above with the injection 
tubule and the reagents injected. The details of this sealing which is 
easy to make, can be inferred from the figure. In the glass vessel there 
is a magnetic stirrer, which can be driven by a magnetic motor. For field-
work a stirrer can be made with the help of a clockwork motor and a second 
magnetic stirrer secured on top, which is independent of electricity. 
Because of the short time necessary for stirring, a single winding of the 
spring is sufficient for each analysis. According to the quantity of 
oxygen expected various concentrations of the leuko-base solution can be 
employed. The MnCl2 and NaOH solutions used hitherto for the Winkler 
method can be adapted, although then both substances are present in a large 
but not disturbingly large excess. The advantage in the use of these 
concentrated solutions is their own small oxygen content. The following 
solutions were used by us for the determination of oxygen. 
1. 40 g MnCl2 4H2O are made up to 100 ml with boiled de-ionised or 
distilled water. 
2. 10 g NaOH are made up in the same way to 100 ml. 
3. 25 g CH3COOH are made up in the same way to 100 ml. 
4. 4 g Leukoberbelin-Blue I are mixed with ca 80 ml boiled water, 0.3 ml 
conc. NH4OH are added and made up to 100 ml. In darkness this solution 
can be kept for longer than 6 months. 
3 
4 
6. Method of determination of Oxygen 
The water sample is flushed through the vessel shown in Fig. 1, 
where, by repeated brief raising of the stopper the vessel becomes free 
of air. After several minutes, when the vessel has been rinsed free of 
the air which has been in contact with the water, the inflow of water is 
stopped up. With the help of the syringe (a separate one is necessary 
for each solution) are then injected one after the other through the silicone 
rubber membrane. 0.5 ml of solution 1 and 0.5 ml of solution 2, are given 
a quick stir with the magnetic stirrer and after ca. 20 secs 0.5 ml of 
solution 3 and 1 ml solution 4 are injected and mixed. Thus is the critical 
part of the determination of oxygen completed. Now the necessary aliquot 
for the filling of the cuvettes is removed with a pipette and the extinction 
measured photometrically. The blue-tinted solution, depending on the 
oxygen content can, however, also be transferred and be measured after a few 
days have elapsed. These conditions are particularly advantageous for 
field-work. The silicone rubber stopper is then rinsed, the remains from 
the previous analysis are poured out of the glass vessel, the stopper is 
replaced and the vessel is flushed with the next water sample to be measured. 
The named quantities of reagent are sufficient for oxygen contents up to 
15 mg O2/l when using a 100 ml reaction vessel. 
7. Evaluation 
The solution of the blue colouring matter obtained is measured in a 
photometer with filter 6l8 nm (Elco II, made by Zeiss) against water. The 
following cuvettes are necessary: 
Range 0-50 µg O2/l cuvettes with path lengths of d = 5 cm 
Range 50-250 µg O2/l cuvettes with path lengths of d = 1 cm 
Range 250-2,500 µg O2/l cuvettes with path lengths of d. = 0.1 cm 
Range 2.5-15 mg O2/l the solution of colouring matter obtained is diluted 
1:10, then path length d = 0.1 cm 
By raising the amount of the leuko-base and giving the corresponding 
dilution of the colouring matter solution formed still higher oxygen contents 
can be measured. We have obtained our calibration curve with Mn(IV) 
suspensions (German Standard Method G2) as we were not successful in 
manufacturing pure aqueous oxygen solutions with O2 concentrations < 300 µg O2/l. 
The quantitative reaction of dissolved oxygen with Mn2+ in an alkaline 
medium is the basis of all Winkler methods, both classical and all modified 
methods. In spite of this the roundabout way via higher manganese hydroxide 
to find the calibration curve cannot be satisfactory. A treatment constant 
of ω = 312 is obtained from the calibration curve. In this way the oxygen 
content per litre of water shows itself. 
a) Extinction x 312 = µg O2/l 
If one bears in mind the dilution of the sample by the added reagents, one 
obtains 
5 
b) Extinction x 312 • = µg O2/l 
Both V and a are in opposition to other Winkler methods and can be implicated 
in the treatment constants. V is found from the size of the reaction vessel 
and ought to lie around 100 ml (magnet stirrer in vessel). a is the constant 
from the treatments and amounts to 2.5 ml. For the case V = 100 ml the 
simplified formula is obtained: 
c) Extinction x 320 = µg O2/l 
Given V 100 ml, the corrected treatment constant must be reckoned 
from b) separately for the reaction vessel used. If less exact results are 
required then the analysis solutions can also be compared with colour 
standards. For the range 2.5 - 15 µg O2/l the result must be multiplied by 
10 on account of the dilution. 
For the range 1 - 10 mg O2/l, single measurements by this method vary 
by 4% from the German Standard Method G2 in which pure water is used. The 
application of this method of oxygen determination in the presence of strongly 
reducing substances such as eg. Fe2+ in conjunction with a semi-automatic 
arrangement working by a different method, which is also suitable for fieldwork, 
is still being worked on. The method is also feasible with sea-water. The 
determination of ozone and H2O2 with leuko-bases of triphenylmethane colouring 
substances is to be published in a later paper. 
Literature 
Gates, E.M., Ellis, G.H.: J. Biol. Chem. 168, 537 (1947) 
Morsches, B., Tolg, G.: Z. analyt. Chem. 250, 8l (1970) 
Perscheid, H., Ballschmiter, K.: Z. analyt. Chem. 258, 15 (1972) 
Yuen, S.H.: Analyst 83, 350 (1958); Z. analyt. Chem. 168, 371 (1959) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Notice 
 
 
Please note that these translations were produced to assist the scientific staff of the 
FBA (Freshwater Biological Association) in their research. These translations were 
done by scientific staff with relevant language skills and not by professional 
translators. 
 
 
 


Determination of dissolved oxygen in water with leukoberbelin-blue I. A quick Winkler method. [Translation from: Z.analyt.Chem. 262 97-99, 1972.]

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Determination of dissolved oxygen in water with leukoberbelin-blue I. A quick Winkler method. [Translation from: Z.analyt.Chem. 262 97-99, 1972.]

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