Regulation (EC) No 648/2004 of the European Parliament and of the Council of 31 March 2004 on detergents (Text with EEA relevance) (c. 648)

ANNEX VIII U.K. TEST METHODS AND ANALYTICAL METHODS

5. Preliminary notes U.K.

5.1. Treatment of samples U.K.

5.1.1. The treatment of non-ionic surface-active agents and formulated detergents prior to the determination of primary biodegradability in the confirmatory test is: U.K.

The purpose of the alcoholic extraction is to eliminate the insoluble and inorganic ingredients of the commercial product, which in some circumstances might upset the biodegradability test.

Products	Treatment
Non-ionic surfactants	None
Formulated detergents	Alcoholic extraction followed by separation of the non-ionic surfactants by ion exchange

Ion-exchange procedure

5.1.2. Isolation and separation of non-ionic surface active agents from soap, anionic and cationic surfactants are required for correct biodegradability tests. U.K.

This is achieved by an ion exchange technique using a macro-porous exchange resin and suitable eluants for fractional elution. Thus soap, anionic and non-ionic surfactants may be isolated in one procedure.

Analytical control

5.1.3. After homogenising, the concentration of anionic and non-ionic surfactants in the detergent is determined according to the MBAS and BiAS analytical procedure. The soap content is determined by a suitable analytical method. U.K.

This analysis of the product is necessary to calculate the quantities required preparing fractions for the biodegradability tests.

Quantitative extraction is not necessary; however, at least 80 % of the non-ionic surfactants should be extracted. Usually, 90 % or more is obtained.

Principle

5.2. From a homogeneous sample (powders, dried paste and dried liquids) an ethanol extract is obtained which contains the surfactants, soap and other alcohol-soluble constituents of the detergent sample. U.K.

The ethanol extract is evaporated to dryness, dissolved in an isopropanol/water mixture and the solution obtained is passed through a strongly acidic cation exchange/macro-porous anion exchange combination heated to 50 ° C. This temperature is necessary to prevent the precipitation of any fatty acids which may be present in acidic media. The non-ionic surfactants are obtained from the effluent by evaporation.

Cationic surfactants, which might upset the degradation test and the analytical procedure, are eliminated by the cation exchanger placed above the anion exchanger.

Chemicals and equipment

5.3. Deionised water U.K.

5.3.1. Ethanol, C₂H₅OH 95 % (v/v) (permissible denaturant: methyl-ethyl ketone or methanol) U.K.

5.3.2. Isopropanol/water mixture (50/50 v/v): U.K.

5.3.3. 50 parts by volume isopropanol, CH₃CHOH.CH₃, and U.K.

• 50 parts by volume water (5.3.1)

• Ammonium bicarbonate solution (60/40 v/v):

5.3.4. 0,3 mol NH₄HCO₃ in 1 000 ml of an isopropanol/water mixture consisting of 60 parts by volume isopropanol and 40 parts by volume water (5.3.1) U.K.

• Cation exchanger (KAT), strongly acidic, resistant to alcohol (50-100 mesh)

5.3.5. Anion exchanger (AAT), macro-porous, Merck Lewatit MP 7080 (70-150 mesh) or equivalent U.K.

5.3.6. Hydrochloric acid, 10 % HCl w/w U.K.

5.3.7. 2 000 ml round-bottomed flask with ground glass stopper and reflux condenser U.K.

5.3.8. 90 mm diameter suction Filter (heatable) for filter papers U.K.

5.3.9. 2 000 ml filter flask U.K.

5.3.10. Exchange columns with heating jacket and tap: inner tube 60 mm in diameter and 450 mm in height (see Figure 4) U.K.

5.3.11. Water-bath U.K.

5.3.12. Vacuum drying oven U.K.

5.3.13. Thermostat U.K.

5.3.14. Rotary evaporator U.K.

5.3.15. Preparation of extract and separation of non-ionic active agents U.K.

5.4. Preparation of extract U.K.

5.4.1. The quantity of surfactant necessary for the degradation test is about 25 g BiAS. U.K.

In preparing extracts for the degradation tests, the quantity of product to be used should be limited to a maximum of 2 000 g. Therefore it may be necessary to carry out the operation two or more times in order to obtain sufficient quantity for the degradation tests.

Experience has shown that there are advantages in using a number of small extractions rather than one large extraction.

Isolation of alcohol-soluble constituents

5.4.2. Add 250 g of the synthetic detergent to be analysed to 1 250 ml ethanol and heat the mixture to boiling point and reflux for one hour with stirring. Pass the hot alcoholic solution through a coarse-pored suction filter heated to 50 ° C and filter rapidly. Wash the flask and suction filter with approximately 200 ml hot ethanol. Collect the filtrate and filter washings in a filter flask. U.K.

In the case of pastes or liquid products to be analysed, make sure that not more than 25 g anionic surfactants and 35 g soap are contained in the sample. Evaporate this weighed sample to dryness. Dissolve the residue in 500 ml ethanol and proceed as described above.

In the case of powders of low apparent density (< 300 g/l) it is recommended to increase the ethanol ratio in the relation 20:1.

Evaporate the ethanolic filtrate to complete dryness, preferably by means of rotary evaporator. Repeat the operation if a greater quantity of extract is required. Dissolve the residue in 5 000 ml isopropanol/water mixture.

5.4.3. Preparation of ion-exchange columns U.K.

CATION-EXCHANGE COLUMN U.K.

Place 600 ml cation-exchange resin (5.3.5) in a 3 000 ml beaker and cover by adding 2 000 ml hydrochloric acid (5.3.7). Allow to stand for at least two hours, with occasional stirring.

Decant the acid and transfer the resin into the column (5.3.11) by means of deionised water. The column should contain a glass-wool plug. Wash the column with deionised water at a rate of 10-30 ml/min until the eluate is free of chloride.

Displace the water with 2 000 ml isopropanol/water mixture (5.3.3) at a rate of 10-30 ml/min. The exchange column is now ready for operation.

ANION-EXCHANGE COLUMN U.K.

Place 600 ml anion-exchange resin (5.3.6) in a beaker and cover by adding 2 000 ml deionised water. Allow the resin to swell for at least two hours. Transfer the resin into the column by means of deionised water. The column should contain a glass-wool plug.

Wash the column with 0,3 M ammonium bicarbonate solution (5.3.4) until free of chloride. This requires about 5 000 ml solution. Wash again with 2 000 ml deionised water.

Displace the water with 2 000 ml isopropanol/water mixture (5.3.3) at a rate of 10-30 ml/min. The exchange column is now in the OH form and ready for operation.

5.4.4. Ion-exchange procedure U.K.

Connect the exchange columns so that the cation-exchange column is placed on top of the anion-exchange column. Heat the exchange columns to 50 ° C using thermostatic control. Heat 5 000 ml of the solution obtained in item 5.4.2 to 60 ° C and pass the solution through the exchanger combination at a rate of 20 ml/min. Wash the columns with 1 000 ml hot isopropanol/water mixture (5.3.3).

To obtain the non-ionic surfactants collect the filtrate and filter washings and evaporate to dryness, preferably by means of a rotary evaporator. The residue contains the BiAS. Add deionised water until a defined volume is obtained and determine the BiAS content in an aliquot. The solution is used as a standard solution of non-ionic surfactants for the degradation test. The solution should be kept at a temperature below 5 ° C.

5.4.5. Regeneration of ion exchange resins U.K.

The cation exchanger is rejected after use.

Passing about 5 000-6 000 ml of ammonium bicarbonate solution (5.3.4) down the column at a flow rate of approximately 10 ml/min until the eluate is free from anionic surfactants (methylene blue test) regenerates the anion-exchange resin. Then pass 2 000 ml isopropanol/water mixture (5.3.3) down the anion exchanger to wash. The anion exchanger is again ready for operation.