Directive 1983/514 - Approximation of the laws of the Member States relating to methods of analysis necessary for checking the composition of cosmetic products - Main contents

EN

Official Journal of the European Communities

L 291/9

determination of dichloromethane and 1,1,1-trichloroethane,

identification and determination of quinolin-8-ol and bis(8-hydroxyquinolinium) sulphate,

determination of ammonia,

identification and determination of nitro-methane,

identification and determination of mercapto-acetic acid in hair-waving, hair-straightening and depilatory products,

identification and determination of hexachlorophene (INN),

determination of tosylchloramide sodium (INN),

determination of total fluorine in dental creams,

identification and determination of organo-mercury compounds,

determination of alkali and alkaline earth sulphides,

4.1.

Chloroform (CHCl3).

4.2.

Carbon tetrachloride (CCl4).

4.3.

Dichloromethane (CH2Cl2).

4.4.

1,1,1-trichloroethane (CH3CCl3).

4.5.

Acetone.

4.6.

Nitrogen.

5.1.

Usual laboratory apparatus.

5.2.

Gas chromatograph fitted with a thermal conductivity detector.

5.3.

Transfer bottle, 50 to 100 ml (see sampling method 5.3) (1).

5.4.

Pressure gas-syringe, 25 or 50 μl (see sampling method 5.4.2.2) (1).

6.1.

Non-pressurized sample: weigh the sample accurately in a stoppered conical flask. Introduce an accurately weighed quantity of chloroform (4.1) as internal standard equivalent to the presumed quantity of dichloromethane and 1,1,1-trichloroethane contained in the sample. Mix throroughly.

6.2.

Pressurized sample: use the sampling method described in the sampling chapter, but with the following refinements:

7.1.

Precolumn

Tubing: stainless steel.

Length: 300 mm

Diameter: 3 or 6 mm.

Packing: same material as used for the analytical column packing.

7.2.

Column

The stationary phase is made of Hallcomid M 18 on chromosorb. The column must yield a resolution ‘R’ equal to, or better than, 1,5, where:

let:

7.3.

As examples the following columns yield the results sought:

Temperature conditions may vary as a function of the apparatus. In the examples they have been set as follows:

=

its response factor,

=

its response factor (made equal to unity),

=

the mass (in grams) of chloroform introduced,

4.1.

Quinolin-8-ol.

4.2.

Benzene. In view of its toxicity great care must be taken when working with benzene.

4.3.

Chloroform.

4.4.

Aqueous sodiun hydroxide, 50 % (m/m) solution.

4.5.

Copper sulphate pentahydrate.

4.6.

Potassium sodium tartrate.

4.7.

M hydrochloric acid.

4.8.

0,5 M sulphuric acid.

4.9.

M sodium hydroxide solution.

4.10.

Ethanol.

4.11.

Butan-1-ol.

4.12.

Glacial acetic acid.

4.13.

0,1 hydrochloric acid.

4.14 ‘Celite 545’or equivalent.

4.15.

Standard solutions

4.15.1.

Weigh 100 mg of quinolin-8-ol (4.1) into a 100 ml standard flask. Dissolve in a little sulphuric acid (4.8) Make up to the mark with sulphuric acid (4.8).

4.15.2.

Weigh 100 mg of quinolin-8-ol into a 100 ml standard flask. Dissolve in ethanol (4.10). Make up to the mark with ethanol (4.10) and mix.

4.16.

Fehling's solution

Solution A

Weigh 7 g of copper sulphate pentahydrate (4.5) into a 100 ml standard flask.

Dissolve in a little water. Make up to the mark with water and mix.

Solution B

Weigh 35 g of potassium sodium tartrate (4.6) into a 100 ml standard flask. Dissolve in 50 ml of water. Add 20 ml of sodium hydroxide (4.4). Make up to the mark with water and mix. Immediately before use, pipette 10 ml of solution A and 10 ml of solution B into a 100 ml standard flask. Make up to the mark and mix.

4.17.

Eluting solvents for thin-layer chromatography

4.18.

2,6-dichloro-4-(chloroin ino)cyclohexa-2,5-dienone, 1 % (m/v) solution in ethanol (4.10).

4.19.

Sodium carbonate, 1 % (m/v) solution in water.

4.20.

Ethanol (4.10), 30 % (v/v) solution in water.

4.21.

Disodium dihydrogen ethylenediaminetetraacetate, 5 % (m/v) solution in water.

4.22.

Buffer solution, pH 7

Weigh 27 g of potassium dihydrogenorthophosphate anhydrous and 70 g of dipotassium hydrogenorthophosphate trihydrate into a one litre standard flask. Make up to the mark with water.

4.23.

Prepared thin-layer plates

Ready made thin-layer plates of a thickness of 0,25 mm (e.g. Merck Kieselgel 60 or equivalent). Before use, spray with 10 ml of reagent (4.21) and dry at 80 oC.

5.1.

100 ml round-bottom flask with ground-glass neck.

5.2.

Standard flasks.

5.3.

Graduated pipettes, 10 and 5 ml.

5.4.

Bulb pipettes, 20, 15, 10 and 5 ml.

5.5.

Separating funnels, 100, 50 and 25 ml.

5.6.

Pleated filter paper, diameter 90 mm.

5.7.

Rotary evaporator.

5.8.

Reflux condenser with ground-glass neck.

5.9.

Spectrophotometer.

5.10.

Optical cells of 10 mm path length.

5.11.

Stirrer hotplate.

5.12.

Glass chromatography column dimensions: 160 mm long with a diameter of 8 mm, a construction at the lower end containing a glass-wool plug, and an adaptor at the upper end for application of pressure.

6.1.1.1.

The pH of part of the test sample is adjusted to 7. 5 and 10 μl are spotted on the starting line of a pretreated silica gel thin-layer plate (4.23).

6.1.1.2.

10 and 30 μl of the standard solution (4.15.2) is spotted on two more points of the starting line after which the plate is developed in one of the two eluents (4.17).

6.1.1.3.

When the solvent front has advanced 150 mm, the plate is dried at 110 oC (for 15 minutes). Under a UV lamp (366 nm) the quinolin-8-ol spots fluoresce yellow.

6.1.1.4.

Spray the plate with sodium carbonate solution (4.19). Dry and spray with 2,6-dichloro-4-(chloroimino)cyclohexa-2,5-dienone solution (4.18). The quinolin-8-ol becomes visible as a blue spot.

6.1.2.1.

Disperse 1 g of the sample in 5 ml of buffer solution (4.22). Then transfer with 10 ml of chloroform (4.3) into a separating funnel and shake. After separation of the chloroform ayer the aqueous layer is extracted twice more with 10 ml of chloroform (4.3) Evaporate the combined and filtered chloroform extracts almost to dryness in a 100 ml round-bottom flask (5.1) on the rotary evaporator (5.7). Dissolve the res due in 2 ml of chloroform (4.3) and spot 10 and 30μl of the solution obtained on a silica gel, thin-layer plate (4.23) in accordance with the method described in 6.1.1.1 onward.

6.1.2.2.

Apply 10 and 30 μl of the standard solution (4.15.2) to the plate and continue as described in 6.1.1.2 to 6.1.1.4.

6.2.1.1.

Weigh 5 g of the sample into a 100 ml round-bottom flask. Add 1 ml of a suphuric acid solution (4.8) and evaporate the mixture almost to dryness under reduced pressure at 50 oC.

6.2.1.2.

Dissolve this residue in 20 ml of warm water. Transfer into a 100 ml standard flask. Rinse three times with 20 ml of water. Make up to 100 ml with water and mix.

6.2.1.3.

Pipette 5 ml of this solution into a 50 ml separating funnel (5.5). Add 10 ml of Fehling's solution (4.16). Extract the quinolin-8-ol copper complex [oxine copper (ISO)] obtained with three times 8 ml of chloroform (4.3).

6.2.1.4.

Filter and collect the chloroform layers in a 25 ml standard flask (5.2). Make up to the mark with chloroform (4.3) and shake. Measure the optical density of the yellow solution against chloroform at 410 nm.

6.2.2.1.

Weigh 0,500 g of the sample into a 100 ml round-bottom flask (4.1). Add 30 ml of benzene (4.2) and 20 ml of hydrochloric acid (4.7). Boil the contents of the flask under reflux, with stirring, for 30 minutes.

6.2.2.2.

Transfer the contents of the flask into a 100 ml separating funnel (5.5). Rinse with 5 ml of 1 N HC1 (4.7). Transfer the aqueous phase into a round-bottom flask (5.1) and wash the benzene phase with 5 ml of hydrochloric acid (4.7).

6.2.2.3.

In the case of emulsions that impede further treatment, mix 0,500 g of the sample with 2 g of Celite 545 (4.14) to form a freely flowing powder. Transfer the mixture in small portions into a glass chromatography column (5.12).

After each addition, tarmp down the column packing. As soon as the whole of the mixture has been transferred into the column, elute with hydrochloric acid (4.13) in such a way that 10 ml of eluate is obtained in approximately 10 minutes (if necessary, this elution can be performed under a slight nitrogen pressure). During the elution it must be ensured that there is always some hydrochloric acid above the column packing. The first 10 ml of eluate is further treated as described in 6.2.2.4.

6.2.2.4.

Evaporate the collectec aqueous phases (6.2.2.2) or the eluate (6.2.2.3) almost to dryness in the rotary evaporator under reduced pressure.

6.2.2.5.

Dissolve the residue in 6 ml of the sodium hydroxide solution (4.9). Add 20 ml of Fehling's solution (4.16) and transfer the contents of the flask into a 50 ml separating funnel (5.5) Rinse the flask with 8 ml of chloroform (4.3). Shake and filter the chloroform phase into a 50 ml standard flask (5.2).

6.2.2.6.

Repeat the extraction three times with 8 ml of chloroform (4.3). Filter the chloroform phases and collect in the 50 ml flask. Make up to the mark with chloroform (4.3) and shake. Measure the optical density of the yellow solution against chloroform (4.3) at 410 nm.

=

milligrams of quinolin-8-ol on the standard curve (7),

=

milligrams of quinolin-8-ol on the standard curve (7),

4.1.

Methanol.

4.2.

Barium chloride dihydrate, 25 % (m/v) solution.

4.3.

Orthoboric acid, 4 % (m/v) solution.

4.4.

Sulphuric acid, 0,25 M standard solution.

4.5.

Anti-foam liquid.

4.6.

Sodium hydroxide, 0,5 M standard solution.

4.7.

Indicator, if required: mix 5 ml of a 0,1 % (m/v) methyl red solution in ethanol with 2 ml of 0,1 % (m/v) methylene blue solution in water.

5.1.

Usual laboratory apparatus.

5.2.

Centrifuge with stoppered 100 ml bottles.

5.3.

Steam distillation apparatus.

5.4.

Potentiometer.

5.5.

Indicating glass electrode and dimercury dichloride (calomel) reference electrode.

6.1.

Weigh into a 100 ml standard flask a mass (m) of the sample corresponding to 150 mg maximum of ammonia.

6.2.

Add 10 ml of water, 10 ml of methanol (4.1) and 10 ml of barium chloride solution (4.2). Make up to 100 ml with methanol (4.1).

6.3.

Mix and leave overnighs in the refrigerator (5 oC).

6.4.

Then filter, or centrifuge the still cold solution in closed tubes for 10 minutes, so as to obtain a clear filtrate or supernatant layer.

6.5.

Pipette 40 ml of this clear solution into the steam distillation apparatus (5.3), followed by 0,5 ml of antifoam liquid (4.5), where appropriate.

6.6.

Distil and collect 200 ml of distillate in a 250 ml beaker containing 10 ml of standard sulphuric acid (4.4) and 0,1 ml of indicator (4.7).

6.7.

Back titrate the excess acid with standard sodium hydroxide solution (4.6).

6.8.

NB: For potentiometric determination, collect 200 ml of distillate in a 250 ml beaker containing 25 ml of orthoboric acid solution (4.3) and titrate with standard sulphuric acid (4.4), recording the neutralization curve.

=

the volume (in inillilitres) of the sodium hydroxide solution (4.6) used,

=

the volume (in millilitres) of the sulphuric acid solution (4.4) used,

4.1.1.

Sodium hydroxide, 0,5 M solution.

4.1.2.

Folin's reagent

Dissolve 0,1 g of sodium 3,4-dihydro-3,4-dioxonaphthalene-l-sulphonate in water and dilute to 100 ml.

5.1.1.

Chloroform (internal standard 1).

5.1.2.

2,4-dimethylheptane (internal standard 2).

5.1.3.

Ethanol, 95 %.

5.1.4.

Nitromethane.

5.1.5.

Chloroform reference solution

Into a tared 25 ml volumetric flask, introduce about 650 mg of chloroform (5.1.1). Accurately reweigh the flask and contents. Make up to 25 ml with 95 % ethanol (5.1.3). Weigh and calculate the percentage by mass of chloroform in this solution.

5.1.6.

2,4-dimethylheptane reference solution

Make up in a similar manner to the chloroform reference solution but weigh 270 mg of 2,4-dimethylheptane (5.1.2) into the 25 ml volumetric flask.

5.2.1.

Gas chromatograph with flame ionization detector.

5.2.2.

Apparatus for sampling of aerosols (transfer bottle, microsyringe connectors, etc.) as described in Chapter II of the Annex to Commission Directive 80/1335/EEC of 22 December 1980 (5).

5.2.3.

Usual laboratory apparatus.

=

retention times (in minutes),

=

its mass (in grams) in the mixture,

=

its response factor,

=

its mass (in grans) in the mixture,

4.1.1.1.

Lead di(acetate) papes.

4.1.1.2.

Hydrochloric acid solution (one volume of concentrated hydrochloric acid plus one volume of water)

4.2.1.1.

Mercaptoacetic acid (thioglycollic acid), 98 % minimum purity assayed by iodometry.

4.2.1.2.

2,2'-dithiodi(acetic acid), 99 % minimum purity assayed by iodometry.

4.2.1.3.

2-mercaptopropionic acid (thiolactic acid), 95 % minimum purity assayed by iodometry.

4.2.1.4.

3-mercaptopropionic acid, 98 % minimum purity assayed by iodometry.

4.2.1.5.

3-mercaptopropane-l,2-diol (1-thioglycerol), 98 % minimum purity assayed by iodometry.

4.2.1.6.

Thin-layer plates, silica gel, ready prepared, 0,25 mm thickness.

4.2.1.7.

Thin-layer plates, aluminium oxide, Merck F 254 E or equivalent.

4.2.1.8.

Hydrochloric acid, concentrated, d4 20 = 1,19 g/ml.

4.2.1.9.

Ethyl acetate.

4.2.1.10.

Chloroform.

4.2.1.11.

Diisopropyl ether

4.2.1.12.

Carbon tetrachloride.

4.2.1.13.

Acetic acid, glacial.

4.2.1.14.

Potassium iodide, 1 % (m/v) solution in water.

4.2.1.15.

Platinum tetrachloride, 0,1 % (m/v) solution in water.

4.2.1.16.1.

Ethyl acetate (4.2.1.9), chloroform (4.2.1.10), diisopropyl ether (4.2.1.11), acetic acid (4.2.1.13) (20: 20: 10: 10, by volume).

4.2.1.16.2.

Chloroform (4.2.1.10), acetic acid (4.2.1.13) (90: 20, by volume).

4.2.1.17.1.

Mix, immediately before use, equal volumes of solution (4.2.1.14) and solution (4.2.1.15).

4.2.1.17.2.

Bromine solution 5 % (m/v):

Dissolve 5 g of bromine in 100 ml of carbon tetrachloride (4.2.1.12).

4.2.1.17.3.

Fluorescein solution, 0,1 % (m/v):

Dissolve 100 mg of fluorescein in 100 ml of ethanol.

4.2.1.17.4.

Hexaammonium heptamolybdate, 10 % (m/v) solution in water.

4.2.1.18.1.

Mercaptoacetic acid (4.2.1.1), 0,4 % (m/v) solution in water.

4.2.1.18.2.

2,2'-dithiodi(acetic) acid (4.2.1.2), 0,4 % (m/v) solution in water.

4.2.1.18.3.

2-mercaptopropionic acid (4.2.1.3), 0,4 % (m/v) solution in water.

4.2.1.18.4.

3-mercaptopropionic acid (4.2.1.4), 0,4 % (m/v) solution in water.

4.2.1.18.5.

3-mercaptopropane-l,2-diol (4.2.1.5), 0,4 % (m/v) solution in water.

the state of activation of the thin layer at the time of chromatographing,

the temperature of the chromatography tank.

=

the mass (in grams) of the test portion,

5.2.2.1.

Mercaptoacetic acid, 98 %.

5.2.2.2.

Hydrochloric acid, d4 10 = 1,19 g/ml.

5.2.2.3.

Methanol.

5.2.2.4.

Cadmium di(acetate) dihydrate, 10 % (m/v) solution in water.

5.2.2.5.

Methyl octanoate, 2 % (m/v) solution in methanol.

5.2.2.6.

Acetate buffer solution (pH 5):

Sodium acetate trihydrate, 77 g.

Acetic acid (glacial), 27,5 g.

Demineralized water to give a final volume of one litre.

5.2.2.7.

Hydrochloric acid, 3 M solution in methanol (5.2.2.3), freshly prepared.

5.2.2.8.

1-methyl-3-nitro 1 -nitrosoguanidine.

5.2.2.9.

Sodium hydroxide, 5 M solution.

5.2.2.10.

Iodine, 0,05 M standard solution.

5.2.2.11.

Diethyl ether.

5.2.2.12.

Diazomethane solution prepared from iV-methyl-AT-nitrosotoluen-4-sulfonamide (Fieser, Reagents for Organic Synthesis (Wiley), 1967)

The solution obtained contains about 1,5 g of diazomethane in 100 ml of diethyl ether. As diazomethane is a toxic and very unstable gas, all experiments must be carried out under a powerful hood and the use of ground-glass apparatus must be avoided (there are special kits for this purpose).

5.2.3.1.

Usual laboratory equipment.

5.2.3.2.

Apparatus for the preparation of diazomethane for in situ methylation (see Fales, H. M., Jaouni, T. M. and Babashak, J. F., Analyt. Chem. 1973, 45, 2302).

5.2.3.3.

Apparatus for the advance preparation of diazomethane (Fieser).

2 ml of methyl octanoate solution (5.2.2.5),

5 ml of hydrochloric acid in methanol (5.2.2.7).

5.2.7.1.

Column

Type: stainless steel.

Length: 2 m.

Diameter: 3 mm.

5.2.7.2.

Packing

20 % didecyl phthalate/chromosorb, WAW 80 to 100 mesh.

5.2.7.3.

Detector

Flame ionization. A suitable sensitivity setting for the electrometer of the flame ionization detector is 8 × 10-10 A.

5.2.7.4.

Gas supplies

Carrier gas: nitrogen.

pressure: 2,2 bar,

flow: 35 ml/min.

Auxiliary gas: hydrogen.

pressure: 1,8 bar,

flow: 15 ml/min.

Detector supplies: as specified by the makers of the apparatus.

5.2.7.5.

Temperature conditions

Injector: 200 oC

Detector: 200 oC

Column: 90 oC

5.2.7.6.

Recorder chart speed

5 mm/min.

5.2.7.7.

Quantity injected

3 μl Carry out five injections.

5.2.7.8.

The conditions of chromatography are given as a guide. They permit the achievement of a resolution ‘R’ equal to, or better than, 1,5, where:

let:

It is recommended that chromatography be terminated by regulating the tempera-ture from 90 to 150 oC at a rate of 10 oC per minute so as to eliminate substances liable to interfere with subsequent measurements.

=

its response factor,

=

its mass (in millegrams) in the mixture,

its response factor,

=

its peak area.

=

its mass (in mill grams) in the mixture,

3.1.

Sulphuric acid, 4 M solution.

3.2.

Celite AW.

3.3.

Ethyl acetate.

3.4.

Eluting solvent: Benzene containing 1 % (v/v) of glacial acetic acid.

3.5.

Visualizing agent I:

Rhodamine B solution: dissolve 100 mg of Rhodamine B in a mixture of 150 ml of diethyl ether, 70 ml of absolute ethanol and 16 ml of water.

3.6.

Visualizing agent II:

2,6-dibromo-4-(cMoroimino)cyclohexa-2,5-dienone solution: dissolve 400 mg of 2,6(dibromo-4-(chloroimino)cyclohexa-2,5-dienone in 100 ml of methanol (prepare fresh daily).

Sodium carbonate solution: dissolve 10 g of sodium carbonate in 100 ml of demineralized water.

3.7.

Reference solution:

Hexachlorophene, 0,05 % (m/v) solution in ethyl acetate.

4.1.

Kiesel gel 254 TLC plates, 200 x 200 mm (or equivalent).

4.2.

Usual TLC equipment.

4.3.

Bath thermostatted at 26 oC to hold the chromatography tank.

5.1.

Thoroughly mix 1 g of homogenized sample with 1 g of Celite AW (3.2) and 1 ml of sulphuric acid (3.1).

5.2.

Dry at 100 oC for two hours.

5.3.

Cool and finely powder the dried residue.

5.4.

Extract twice with 10 ml of ethyl acetate (3.3) each time, centrifuge after each extraction and combine the ethyl acetate layers.

5.5.

Evaporate at 60 oC.

5.6.

Dissolve the residue in 2 ml of ethyl acetate (3.3).

6.1.

Place 2 µl of the test sample solution (5.6) and 2 µl of the reference solution (3.7) on a TLC plate (4.1).

6.2.

Saturate the tank (4.3) with the eluting solvent (3.4).

6.3.

Place the TLC plate in the tank and elute up to 150 mm.

6.4.

Remove the TLC plate and dry in a ventilated oven at a temperature of about 105 oC.

6.5.

Visualization

Hexachlorophene spots on the thin-layer plate are visualized as indicated under 6.5.1 or 6.5.2.

6.5.1.

Spray the visualizing agent I (3.5) evenly on the plate. After 30 minutes examine the plate under UV light at 254 nm.

6.5.2.

Spray the 2,6-dibromo-4-(chloroimino)cyclohexa-2,5-dienone solution of visualizing agent II (3.6) evenly on the plate. Subsequently spray the plate with sodium carbonate solution (3.6). Examine the plate in daylight after 10 minutes drying at room temperature.

7.1.

Visualizing agent I (3.5):

Hexachlorophene is revealed as a bluish spot on a yellow-orange fluorescent background and has an Rf of approximately 0,5.

7.2.

Visualizing agent II (3.6):

Hexachlorophene is revealed as a sky-blue to turquoise coloured spot on a white background and has an Rf of approximately 0,5.

4.1.

Ethyl acetate.

4.2.

N -methyl- N -nitroso-p toluenesulphonamide (diazald).

4.3.

Diethyl ether.

4.4.

Methanol.

4.5.

2-(2-ethoxyethoxy)ethanol (carbitol).

4.6.

Formic acid.

4.7.

Potassium hydroxide, 50 % (m/m) aqueous solution (prepare fresh daily).

4.8.

Hexane for spectroscopy.

4.9.

Bromochlorophene (standard No 1).

4.10.

4,4',6,6'-tetrachloro-2,2'-thiodiphenol (standard No 2).

4.11.

2,4,4'-trichloro- 2-hydroxy-diphenyl ether (standard No 3).

4.12.

Acetone.

4.13.

4 M sulphuric acid.

4.14.

Celite AW.

4.15.

Formic acid/ethyl acetate, 10 % (v/v) solution.

4.16.

Hexachlorophene.

5.1.

Usual laboratory glassware.

5.2.

Mini-apparatus for the preparation of diazomethane (Analyt. Chem., 1973, 45, 2302-2).

5.3.

Gas chromatograph equipped with a 63 Ni source electron capture detector.

6.1.1.

Accurately weigh about 50 mg of standard No 1, 2 or 3 (4.9, 4.10 or 4.11) and 50 mg of hexachlorophene (4.16) into a 100 ml volumetric flask. Make up to volume with ethyl acetate (4.1) (solution A). Dilute 10 ml of solution A to 100 ml with ethyl acetate (4.1) (solution B).

6.1.2.

Accurately weigh about 50 mg of standard No 1, 2 or 3 (4.9, 4.10 or 4.11) into a 100 ml volumetric flask. Make up to volume with ethyl acetate (4.1) (solution C ).

=

retention times (in minutes),

:

280 oC,

:

2,3 bar.

=

the hexachlorophene,

=

the hexachlorophene,

4.6.1.

1-butanol /ethanol (4.5) /water (40: 4: 9; v/v/v), or

4.6.2.

Chloroform /acetone (6: 4; v/v).

5.1.

Normal laboratory apparatus.

5.2.

Usual thin-layer chromatography equipment.

5.3.

Photodensitometer.

6.2.1.

Take 30 ml of the filtrate and extract three times with 15 ml of diethyl ether (4.4). If necessary dry the ethereal phases and collect them in a 50 ml graduated flask. Make up with diethyl ether (4.4).

6.2.2.

Take 25 ml of the dried ethereal extract and evaporate to dryness in a nitrogen stream. Redissolve the residue with 1 ml of ethanol (4.5).

6.3.1.

Spot 20 μl of the ethanolic residue (6.2) on to a thin-layer chromatography plate (4.7).

At the same time and in the same manner, apply 8, 12, 16 and 20 μl of the standard solution of 4-toluenesulphonamide (4.2).

6.3.2.

Then allow to develop approximately 150 mm in the development solvent (4.6.1 or 4.6.2).

6.3.3.

After completely evaporating the development solvent, place the plate for two to three minutes in an atmosphere of chlorine vapour, which is produced by pouring about 100 ml of hydrochloric acid (4.9) over about 2 g of potassium permanganate (4.8) in a closed vessel. Remove the excess chlorine by heating the plate to 100 oC for five minutes. Then spray the plate with the reagent (4.10).

=

the 4-toluenesulphonamide-chloramine-T conversion factor,

4.7.1.

Stock solution, 0,250 mg F-/ml. Weigh accurately 138,1 mg of sodium fluoride (4.1) and dissolve in water (4.2). Quantitatively transfer the solution into a 250 ml volumetric flask (5.5). Dilute to the mark with water (4.2) and mix.

4.7.2.

Diluted stock solution, 0,050 mg F-/ml. Transfer by pipette 20 ml of the stock solution (4.7.1) into a 100 ml volumetric flask (5.5). Dilute to the mark with water and mix.

5.1.

Standard laboratory equipment.

5.2.

Gas chromatograph fitted with a flame ionization detector.

5.3.

Vortex swirl mixer or equivalent.

5.4.

Bühler, shaker, type SMB1 or equivalent.

5.5.

Volumetric flasks, 100 and 250 ml, made of polypropylene.

5.6.

Centrifuge tubes (glass); 20 ml with teflon lined screw-caps, Sovirel type 611-56 or equivalent. Clean tubes and screw-caps by leaching several hours in perchloric acid (4.11), followed by five subsequent rinsings with water (4.2), and finally dry at 100 oC.

5.7.

Pipettes, adjustable to deliver volumes of 50 to 200 μl, with disposable plastics tips.

5.8.

Distillation apparatus, fitted with a three-ball Schneider column or an equivalent Vigreux column.

6.1.1.

Select a dental-cream tube not previously opened, cut open the tube and remove the whole contents. Transfer to a plastics container, mix thoroughly and store under conditions avoiding deterioration.

6.1.2.

Weigh accurately 150 mg (m) of sample into a centrifuge tube (5.6), add 5 ml of water (4.2) and homogenize (5.3);

6.1.3.

Add 1 ml of xylene (4.5).

6.1.4.

Add dropwise 5 ml of hydrochloric acid (4.3) and homogenize (5.3).

6.1.5.

Add, by pipette, 0,5 ml of chlorotriethylsilane/internal standard solution (4.9) into the centrifuge tube (5.6).

6.1.6.

Close the tube with the screw-cap (5.6) and mix for 45 minutes thoroughly on a shaker (5.4) set at 150 strokes per minute.

6.1.7.

Centrifuge 10 minutes at such a speed as to produce a clear separation of the phases, uncap the tube, withdraw the organic layer and inject 3 μl of the organic phase on to the column of the gas chromatograph (5.2).

Remark:

It takes about 20 minutes before all components are eluted.

6.1.8.

Repeat the injection, calculate the average peak area ratio (ATEFS/ACH) and read the corresponding amount of fluorine (in milligrams (m1)) from the calibration graph (6.3).

6.1.9.

Calculate the total fluorine content of the sample (in per cent by mass of fluorine) as indicated in paragraph 7.

6.2.1.

Column: stainless steel.

Length: 1,8 m.

Diameter: 3 mm.

Support: Gaschrom Q 80 to 100 mesh.

Stationary phase: silicon oil DC 200 or equivalent, 20 %. Condition the column overnight at 100 oC (carrier gas flow at 25 ml nitrogen per minute) and repeat every night. After each fourth or fifth injection recondition the column by heating for 30 minutes at 100 oC.

Temperatures:

Gas flow carrier: 35 ml of nitrogen per minute.

6.3.1.

Place, by pipette, into a series of six centrifuge tubes (5.6), 0, 1, 2, 3, 4 and 5 ml of the diluted fluoride standard solution (4.7.2). Make up the volume in each tube to 5 ml with water (4.2).

6.3.2.

Proceed as described under 6.1.3 to 6.1.6 inclusive.

6.3.3.

Inject 3 μl of the organic phase on to the column of the gas chromatograph (5.2).

6.3.4.

Repeat the injection and calculate the average peak ratio (ATEFS/ACH).

6.3.5.

Plot a calibration graph correlating the mass of fluorine (in milligrams) in the standard solutions (6.3.1) and the peak area ratio (ATEFS/ACH) measured under 6.3.4. Connect the points of the graph with the best fitting straight line calculated by regression analysis.

=

the test portion (in milligrams) (6.1.2),

2.1.

Sulphuric acid, 25 % (v/v).

2.2.

l,5-diphenyl-3-thiocarbazone (dithizone): 0,8 mg in 100 ml carbon tetrachloride (2.4).

2.3.

Nitrogen.

2.4.

Carbon tetrachloride.

2.5.

Development solvent: hexane /acetone, 90:10 (v/v).

2.6.

Standard solution, 0,001 % in water of:

sodium 2-(ethylmercuriothio)benzoate,

ethylmercury chloride or methylmercury chloride,

phenylmercury nitrate or phenylmercury acetate,

mercury dichloride or mercury di(acetate).

2.7.

Ready prepared silica gel plates (e.g. Merck 5721 or equivalent).

2.8.

Sodium chloride.

3.1.

Normal laboratory equipment.

3.2.

Normal TLC apparatus.

3.3.

Phase-separating filter.

4.1.1.

Dilute 1 g of sample in a centrifuge tube by titration with 20 ml of distilled water. Obtain the maximum dispersion and warm to 60 oC in a water bath. Add 4 g of sodium chloride (2.8). Shake. Allow to cool.

4.1.2.

Centrifuge for at least 20 minutes at 4 500 rev/min in order to separate the greater part of the solid from the liquid. Filter into a separating funnel and add 0,25 ml of sulphuric acid solution (2.1).

4.1.3.

Extract several times with 2 or 3 ml of dithizone solution (2.2) until the last organic phase remains green.

4.1.4.

Filter each organic phase sequentially through a phase-separating filter (3.3).

4.1.5.

Evaporate to dryness in a stream of nitrogen (2.3).

4.1.6.

Dissolve with 0,5 ml of carbon tetrachloride (2.4). Apply this solution immediately as indicated in 4.2.1.

4.2.1.

Apply immediately 50 μl of the carbon tetrachloride solution obtained in 4.1.6 on to a silica gel plate (2.7). Treat simultaneously 10 ml of standard solution (2.6) as in 4.1 and apply 50 μl of the solution obtained in 4.1.6 on the same plate.

4.2.2.

Place the plate in the solvent (2.5) and allow the latter to rise 150 mm. The organomercury compounds appear as coloured spots whose colour is stable, provided the plate is covered by a glass plate immediately the solvent evaporates.

For example, the following Rf values are obtained:

3.1.

Concentrated nitric acid, d4 20 = 1,41 g/ml.

3.2.

Concentrated sulphuric acid, d4 20 = 1,84 g/ml.

3.3.

Redistilled water.

3.4.

Potassium permanganate, 7 % (m/v) solution.

3.5.

Hydroxylammonium chloride, 1,5 % (m/v) solution.

3.6.

Dipotassium peroxodisulphate, 5 % (m/v) solution.

3.7.

Tin dichloride, 10 % (m/v) solution.

3.8.

Concentrated hydrochloric acid, d4 20 = 1,18 g/ml.

3.9.

Palladium dichloride impregnated glass wool, 1 % (m/m).

4.1.

Normal laboratory equipment.

4.2.

Apparatus for flameless atomic absorption mercury determination (cold vapour technique), including the necessary glassware. Path length of the cell at least 100 mm.

5.1.1.

Weigh accurately 150 mg of the sample (m). Add 10 ml of nitric acid (3.1) and leave to digest for three hours in an airtight flask in a water bath at 55 oC, shaking at regular intervals. At the same time, carry out a blank test on the reagents.

5.1.2.

After cooling, add 10 ml of sulphuric acid (3.2) and return to the water bath at 55 oC for 30 minutes.

5.1.3.

Place the flask in an ice bath and add carefully 20 ml of water (3.3).

5.1.4.

Adding 2 ml aliquots of 7 % potassium permanganate solution (3.4) until the solution remains coloured. Return to the water bath at 55 oC for a further 15 minutes.

5.1.5.

Add 4 ml of dipotassium peroxodisulphate solution (3.6). Continue to warm in the water bath at 55 oC for 30 minutes.

5.1.6.

Allow to cool and transfer the contents of the flask into a 100 ml standard flask. Rinse the flask with 5 ml of hydroxylammonium chloride (3.5) and then rinse four times with 10 ml of water (3.3). The solution should be completely decolorized. Make up to the mark with water (3.3).

5.2.1.

Place 10 ml of the test solution (5.1.6) in the glass vessel for the cold vapour mercury determination (4.2). Dilute with 100 ml of water (3.3) and subsequently 5 ml of sulphuric acid (3.2) and 5 ml of tin dichloride solution (3.7). Mix after each addition. Wait 30 seconds to reduce all ionic mercury to the metallic state and take a reading (n).

5.2.2.

Place some palladium dichloride impregnated glass wool (3.9) between the mercury reduction vessel and the flow cell of the instrument (4.2). Repeat 5.2.1 and record the reading. If the reading is not zero mineralization was incomplete and analysis must be repeated.

=

the mass (in milligrams) of the test sample.

7.1.

To improve mineralization it might be necessary to start by diluting the sample.

7.2.

If absorption of the mercury by the substrate is suspected, a quantitative determination by the method of standard additions should be done.

4.1.

Concentrated hydrochloric acid, d4 20 = 1,19 g/ml.

4.2.

Sodium thiosulphate, 0,1 M standard solution.

4.3.

Iodine, 0,05 M standard solution.

4.4.

Disodium sulphide.

4.5.

Cadmium di(acetate).

4.6.

Concentrated ammonia, d4>20 = 0,90 g/ml.

4.7.

Ammoniacal solution of cadmium di(acetate): dissolve 10 g of cadmium di(acetate) (4.5) in approximately 50 ml of water. Add ammonia (4.6) until the precipitate redissolves (i.e. approximately 20 ml). Make up to the 100 ml mark with water.

4.8.

Nitrogen.

4.9.

Solution of ammonia M.

5.1.

Usual laboratory equipment.

5.2. 100 ml round-bottom flask with three standard ground-glass necks.

5.3. Two 150 ml conical flasks with ground-glass necks, fitted with a device comprising a dip tube and a side outlet tube for releasing the entraining gas.

5.4. One long-stem tunnel.

6.1.1.

Take a package which has not been previously opened. Weigh accurately a mass (m) (expressed in grams) of the product corresponding to not more than 30 mg of sulphide ions in the round-bottom flask (5.2). Add 60 ml of water and two drops of an anti-foaming liquid.

6.1.2.

Transfer 50 ml of solution (4.7) to each of the two conical flasks (5.3).

6.1.3.

Fit a dropping funnel, the dip tube and the outlet tube on to the round-bottom flask (5.2). Connect the outlet tube to the conical flasks (5.3) set up in series by means of PVC tubing.

NB: The entraining apparatus must pass the following leak-tightness test: simulating the test conditions, replace the product to be determined by 10 ml of a sulphide solution (prepared from 4.4) containing ‘X mg’ of sulphide (iodometrically determined). Let ‘Y’ be the number of milligrams of sulphide found at the end of this operation. The difference between quantity ‘X’ and quantity ‘Y’ must not exceed 3 %.

6.1.4.

Pass nitrogen (4.8) through for 15 minutes, at a rate of two bubbles per second, in order to expel the air contained in the round-bottom flask (5.2).

6.1.5.

Heat the round-bottom flask to 85 ± 5 oC.

6.1.6.

Stop the nitrogen (4.8) stream and add 40 ml of hydrochloric acid (4.1) drop by drop.

6.1.7.

Turn the nitrogen (4.8) stream on again when nearly all the acid has been transferred, leaving a minimum liquid seal to prevent leakage of hydrogen sulphide.

6.1.8.

Cease heating after 30 minutes. Allow the flask (5.2) to cool and continue to pass the nitrogen (4.8) stream through for at least one-and-a-half hours.

6.2.1.

Filter the cadmium sulphide through a long-stem funnel (5.4).

6.2.2.

Rinse the conical flasks (5.3) first with the ammonia solution (4.9) and pour on the filter. Then rinse with distilled water and use the water to wash the precipitate retained by the filter.

6.2.3.

Complete the washing of the precipitate with 100 ml of water.

6.2.4.

Place the paper filter in the first conical flask that contained the precipitate. Add 25 ml (n1) of the iodine solution (4.3), approximately 20 ml of hydrochloric acid (4.1) and 50 ml of distilled water.

6.2.5.

Determine the excess iodine using the sodium thiosulphate solution (n2) (4.2).

=

the number (in millilitres) of iodine standard solution (4.3) used,