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ANNEX VIIIU.K.TEST METHODS AND ANALYTICAL METHODS
3.Determination of non-ionic surfactants in biodegradation test liquorsU.K.
3.3.MethodU.K.
3.3.1.Concentration and separation of the surfactantU.K.
Filter the aqueous sample through a qualitative filter paper. Discard the first 100 ml of the filtrate.
Into the stripping apparatus, previously rinsed with ethyl acetate, place a measured quantity of the sample, such that it contains between 250-800 g non-ionic surfactant.
To improve the separation add 100 g sodium chloride and 5 g sodium bicarbonate.
If the volume of the sample exceeds 500 ml, add these salts to the stripping apparatus in solid form, and dissolve by passing nitrogen or air through.
If a smaller-sized sample is used, dissolve the salts in 400 ml water and then add to the stripping apparatus.
Add water to bring the level to the upper stopcock.
Cautiously add 100 ml ethyl acetate on top of the water.
Fill the wash-bottle in the gas-line (nitrogen or air) two-thirds full with ethyl acetate.
Pass a gas stream of 30-60 l/h through the apparatus; the use of a flowmeter is recommended. The rate of aeration must be increased gradually at the beginning. The gas rate must be so adjusted that the phases remain noticeably separate to minimise the mixing of the phases and the solution of the ethyl acetate in the water. Stop the gas flow after five minutes.
If there is a reduction of more than 20 % in the volume of the organic phase through solution in water, the sublation must be repeated paying special attention to the rate of gas flow.
Run off the organic phase into a separating funnel. Return any water in the separating funnel from the aqueous phase — it should only be a few ml — to the stripping apparatus. Filter the ethyl acetate phase through a dry qualitative filter paper into a 250 ml beaker.
Put a further 100 ml ethyl acetate into the stripping apparatus and again pass nitrogen or air through for five minutes. Draw off the organic phase into the separating funnel used for the first separation, reject the aqueous phase and run the organic phase through the same filter as the first ethyl acetate portion. Rinse both the separating funnel and the filter with about 20 ml ethyl acetate.
Evaporate the ethyl acetate extract to dryness using a water-bath (fume cupboard). Direct a gentle stream of air over the surface of the solution to accelerate the evaporation.
3.3.2.Precipitation and filtrationU.K.
Dissolve the dry residue from 3.3.1 in 5 ml methanol, add 40 ml water and 0,5 ml dilute HCl (3.2.3) and stir the mixture with a magnetic stirrer.
To this solution add 30 ml of precipitating agent (3.2.6) from a measuring cylinder. The precipitate forms after repeated stirring. After stirring for ten minutes leave the mixture to stand for at least five minutes.
Filter the mixture through a Gooch crucible, the base of which is covered with a glass-fibre filter paper. First wash the filter under suction with about 2 ml glacial acetic acid. Then thoroughly wash the beaker, magnet, and crucible with glacial acetic acid, of which about 40-50 ml is necessary. It is not necessary to quantitatively transfer the precipitate adhering to the sides of the beaker, to the filter, because the solution of the precipitate for the titration is returned to the precipitating beaker, and the remaining precipitate will then be dissolved.
3.3.3.Dissolution of the precipitateU.K.
Dissolve the precipitate in the filter crucible by the addition of hot ammonium tartrate solution (about 80 ° C) (3.2.8) in three portions of 10 ml each. Allow each portion to stand in the crucible for some minutes before being sucked through the filter into the flask.
Put the contents of the filter flask into the beaker used for the precipitation. Rinse the sides of the beaker with a further 20 ml of tartrate solution to dissolve the rest of the precipitate.
Carefully wash the crucible, adapter and filter flask with 150-200 ml water, and return the rinsing water to the beaker used for the precipitation.
3.3.4.The titrationU.K.
Stir the solution using a magnetic stirrer (3.2.16), add a few drops of bromocresol purple (3.2.5) and add the dilute ammonia solution (3.2.9) until the colour turns violet (the solution is initially weakly acid from the residue of acetic acid used for rinsing).
Then add 10 ml standard acetate buffer (3.2.10), immerse the electrodes in the solution, and titrate potentiometrically with standard ‘carbate solution’ (3.2.11), the burette tip being immersed in the solution.
The titration rate should not exceed 2 ml/min.
The endpoint is the intersection of the tangents to the two branches of the potential curve.
It will be observed occasionally that the inflection in the potential curve becomes flattened; this can be eliminated by carefully cleaning the platinum electrode (by polishing with emery paper).
3.3.5.Blank determinationsU.K.
At the same time run a blank determination through the whole procedure with 5 ml methanol and 40 ml water, according to the instructions in 3.3.2. The blank titration should be below 1 ml, otherwise the purity of the reagents (3.2.3, 3.2.7, 3.2.8, 3.2.9, 3.2.10) is suspect, especially their content of heavy metals, and they must be replaced. The blank must be taken into account in the calculation of the results.
3.3.6.Control of the factor of the ‘carbate solution’U.K.
Determine the factor for the carbate solution on the day of use. To do this, titrate 10 ml of the copper sulphate solution (3.2.12) with ‘carbate solution’ after the addition of 100 ml water and 10 ml standard acetate buffer (3.2.10). If the amount used is a ml, the factor f is:
and all the results of the titration are multiplied by this factor.