1982L0434 — EN — 10.04.1990 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B SECOND COMMISSION DIRECTIVE
of 14 May 1982
on the approximation of the laws of the Member States relating to methods of analysis necessary
for checking the composition of cosmetic products
(82/434/EEC)
(OJ L 185, 30.6.1982, p. 1)
Amended by:
Official Journal
No page date
►M1Commission Directive 90/207/EEC of 4 April 1990 L 108 92 28.4.1990
▼B
SECOND COMMISSION DIRECTIVE
of 14 May 1982
on the approximation of the laws of the Member States relating to
methods of analysis necessary for checking the composition of
cosmetic products
(82/434/EEC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,
Having regard to the Treaty establishing the European Economic
Community,
Having regard to Council Directive 76/768/EEC of 27 July 1976 on the
approximation of the laws of the Member States relating to cosmetic
products(1), as amended by Directive 79/661/EEC(2), and in particular
Article 8 (1) thereof,
Whereas Directive 76/768/EEC provides for the official testing of
cosmetic products with the aim of ensuring that the conditions laiddown by Community provisions concerning the composition ofcosmetic products are satisfied;
Whereas all the necessary methods of analysis should be drawn up as
quickly as possible; whereas, the first step towards the attainment ofthis objective having already been taken by the definition of certain
methods in Commission Directive 80/1335/EEC(
3), the second step is
to consist in the definition of methods for identification of someoxidizing agents and determination of hydrogen peroxide in cosmetichair-care products, identification and semi-quantitative determinationof certain oxidation colorants in hair dyes, identification and determi-
nation of nitrite, identification and determination of free
formaldehyde, determination of resorcinol in shampoos and hairlotions, and determination of methanol in relation to ethanol orpropan-2-ol;
Whereas the measures laid down in this Directive are in accordance
with the opinion of the Committee on the adaptation of Directive 76/768/EEC to technical progress,
HAS ADOPTED THIS DIRECTIVE:
Article 1
Member States shall take all necessary steps to ensure that, during offi-
cial testing of cosmetic products:
— identification of oxidizing agents and determination of hydrogen
peroxide in hair-care products,
— identification and semi-quantitative determination of certain oxida-
tion colorants in hair dyes,
— identification and determination of nitrite,
— identification and determination of free formaldehyde,
— determination of resorcinol in shampoos and hair lotion,— determination of methanol in relation to ethanol or propan-2-ol
are performed in accordance with the methods described in the Annex.
Article 2
Member States shall bring into force the laws, regulations or adminis-
trative provisions necessary to comply with this Directive not later than
31 December 1983.
They shall forthwith inform the Commission thereof.1982L0434 — EN — 10.04.1990 — 001.001 — 2
(1) OJ No L 262, 27. 9. 1976, p. 169.
(2) OJ No L 192, 31. 7. 1979, p. 35.
(3) OJ No L 383, 31. 12. 1980, p. 27.
▼B
Article 3
This Directive is addressed to the Member States.1982L0434 — EN — 10.04.1990 — 001.001 — 3
▼B
ANNEX
I.IDENTIFICATION OF OXIDIZING AGENTS AND DETERMINA-
TION OF HYDROGEN PEROXIDE IN HAIR-CARE PRODUCTS
PURPOSE AND SCOPE
The iodometric determination of hydrogen peroxide in cosmetics is only
possible in the absence of other oxidizing agents that form iodine from iodides.
Consequently, before the iodometric determination of hydrogen peroxide it is
necessary to detect and identify any other oxidizing agents present. This identi-fication breaks down into two stages; the first covers the persulphates, the
bromates and hydrogen peroxide and the second covers barium peroxide.
A. IDENTIFICATION OF PERSULPHATES, BROMATES AND
HYDROGEN PEROXIDE
1. PRINCIPLE
Sodium persulphate, potassium persulphate and ammonium persulphate;
potassium bromate, sodium bromate and hydrogen peroxide — whether
or not originating from barium peroxide — are identified by means of
descending paper chromatography, use being made of two developing
solvent.
2. REAGENTS
All reagents should be of analytical purity.
2.1. 0·5 % (m/v) aqueous reference solutions of the following compounds:2.1.1. Sodium persulphate2.1.2. Potassium persulphate2.1.3. Ammonium persulphate2.1.4. Potassium bromate2.1.5. Sodium bromate2.1.6. Hydrogen peroxide2.2. Developing solvent A, 80 % (v/v) ethanol2.3. Developing solvent B, benzene — methanol — 3-methyl butan-1-ol —
water (34:38:18:10 by vol)
2.4. Detecting agent A, 10 % (m/v) aqueous solution of potassium iodide2.5. Detecting agent B, 1 % (m/v) aqueous solution of starch
2.6. Detecting agent C, 10 % (m/m) hydrochloric acid
2.7. 4N hydrochloric acid
3. APPARATUS AND EQUIPMENT
3.1. Chromatography paper (Whatman paper No 3 and No 4 or their equiva-
lents)
3.2. Micropipette, 1 µl3.3. Standard flasks, 100 ml3.4. Fluted filters3.5. Apparatus for descending paper chromatography
4. SAMPLE PREPARATION
4.1. Water soluble products
Make two solutions of eachsample by dissolving 1 g and 5 g of th e
product respectively in 100 ml of water. Use 1 µl of eachof th ese solu-
tions for carrying out the paper chromatography described in Section 5.1982L0434 — EN — 10.04.1990 — 001.001 — 4
▼B
4.2. Products sparingly soluble in water
4.2.1. Weighout 1 g and 5 g of th e sample and disperse in 50 ml of water,
make up to 100 ml withwater in eachcase and mix. Filter th e two
dispersions over a fluted filter (3.4) and use 1 µl of eachof th e filtrates
in order to carry out the paper chromatography described in Section 5.
4.2.2. Prepare once again two dispersions of eachsample by dispersing 1 g
and 5 g in 50 ml of water, acidify with dilute hydrochloric acid (2.7),
make up with water to 100 ml and mix. Filter the dispersions through a
fluted filter (3.4) and use 1 µl of the two filtrates in order to carry out
the paper chromatography described in Section 5.
4.3. Creams
Disperse 5 g and 20 g of eachproduct in 100 ml of water and use th e
dispersions to carry out the paper chromatography described in Section
5.
5. METHOD
5.1. Put an appropriate quantity of solvents A (2.2) and B (2.3) into two
separate chromatography tanks in order to carry out the descending
paper chromatography. Saturate the chromatography tanks for at least
24 hours with solvent vapour.
5.2. Apply 1 µl of one sample solution and of one reference solution
prepared according to Sections 4 and 2.1 to eachstarting point on a
strip of chromatography paper (Whatman No 3 or equivalent) 40 cm
long and 20 cm wide (3.1) or another suitable format and evaporate
the solution in air.
5.3. Place the chromatography strip (5.2) in the chromatography tank filled
with developing solvent A (5.1) and develop until the solvent front has
advanced 35 cm (about 15 hours).
5.4. Repeat the procedure described in Sections 5.2 and 5.3, using chroma-
tography paper (Whatman No 4 or equivalent) (3.1) and developing
solvent B. Chromatograph until the solvent front has advanced 35 cm
(about five hours).
5.5. After development remove the chromatograms and dry them in air.
5.6. Reveal the spots in the chromatogram by spraying it successively with:
5.6.1. detecting agent A (2.4) followed shortly thereafter by detecting agent B
(2.5). The spots of the persulphates will now appear first in the chroma-
togram and will be followed by the hydrogen peroxide spots. Mark the
spots witha pencil;
5.6.2. detecting agent C (2.6) on the chromatograms obtained in accordance
withSection 5.6.1; th e presence of bromates will now be indicated by
greyish blue spots in the chromatogram.
5.7. Under the abovementioned conditions pertaining to developing solvents
A (2.2) and B (2.3), the Rf values of the reference substances (2.1) are
approximately as follows:
Developing solvent
A (2.2)Developing solvent
B (2.3)
Sodium persulphate 0·40 0·10
Potassium persulphate 0·40 0·02 + 0·05Ammonium persul-
phate0·50 0·10 + 0·20
Sodium bromate 0·40 0·20
Potassium bromate 0·40 0·10 + 0·20
Hydrogen peroxide 0·80 0·801982L0434 — EN — 10.04.1990 — 001.001 — 5
▼B
B. IDENTIFICATION OF BARIUM PEROXIDE
1. PRINCIPLE
Barium peroxide is identified by the formation of hydrogen peroxide
after acidification of the sample (A.4.2), and by the presence of the
barium ion:
— in the absence of persulphates (A), by adding dilute sulphuric acid
to a portion of the acid sample solution (B.4.1) as a result of which
a white precipitate of barium sulphate is formed. The presence of
barium ions in the sample (B.4.1) is again confirmed by paper chro-
matography in the manner described below (B.5),
— where barium peroxide and persulphates are present simultaneously
(B.4.2) by digesting the residue from the solution (B.4.2) in an
alkali; after dissolution in hydrochloric acid, the presence of barium
ions is confirmed in the solution of the melt (B.4.2.3) by paper
chromatography and/or by barium sulphate precipitation.
2. REAGENTS
2.1. Methanol2.2. 36 % (m/m) concentrated hydrochloric acid2.3. 6N hydrochloric acid2.4. 4N sulphuric acid2.5. Rhodizonic acid disodium salt2.6. Barium chloride (BaCl
2·2 H2O)
2.7. Anhydrous sodium carbonate
2.8. 1 % (m/v) aqueous solution of barium chloride2.9. Developing solvent consisting of methanol, concentrated hydrochloric
acid (concentration 36 %) and water (80: 10: 10 by vol)
2.10. Detecting agent, 0·1 % (m/v) aqueous solution of rhodizonic acid diso-
dium salt, to be freshly prepared immediately before use.
3. APPARATUS AND EQUIPMENT
3.1. Micropipette, 5 µl
3.2. Platinum crucibles
3.3. Standard flasks, 100 ml3.4. Chromatography paper Schleicher and Schull 2043 b or equivalent.
Clean the paper by developing it overnight in a descending chromato-
graphy tank (A.3.5) containing developing solvent (B.2.9) and then dry.
3.5. Fluted filter paper3.6. The usual apparatus for carrying out ascending paper chromatography
4. SAMPLE PREPARATION
4.1. Products in which persulphate are absent
4.1.1. Disperse 2 g of the product in 50 ml of water and bring the pH of the
dispersion to about 1 with hydrochloric acid (B.2.3)
4.1.2. Transfer the dispersion with water into a 100-ml standard flask, make
up to the mark with water and mix. Use this dispersion for the paper
chromatography analysis described in Section 5 and for identification
of barium by precipitation of the sulphate.
4.2. Products in which persulphates are present
4.2.1. Disperse 2 g of the product in 100 ml of water and filter.
4.2.2. Add to the dried residue seven to 10 times its weight of sodium carbo-
nate (B.2.7), mix and melt the mixture in a platinum crucible (B.3.2)
for half an hour.1982L0434 — EN — 10.04.1990 — 001.001 — 6
▼B
4.2.3. Cool to room temperature, dissolve the melt in 50 ml of water and filter
(B.3.5)
4.2.4. Dissolve the residue from the melt in hydrochloric acid (B.2.3) and
make up to 100 ml with water. Use this solution for the paper chroma-
tography analysis described in Section 5 and for the identification of
barium by precipitation of the sulphate.
5. METHOD
5.1. Place an appropriate quantity of developing solvent (B.2.9) in a tank for
ascending paper chromatography and saturate the tank for at least 15
hours.
5.2. On a piece of chromatography paper — pretreated as described in
Section B.3.4 — apply 5 µl of eachof th e solutions prepared in accor-
dance withSections B.4.1.2 and B.4.2.4 and reference solution B.2.8 at
three starting points.
5.3. Dry the sample and reference spots in air. Develop the chromatogram
until the solvent front has ascended 30 cm.
5.4. Remove the chromatogram from the tank and dry in air.5.5. Reveal the spots on the chromatogram by spraying the paper with
detecting agent B.2.10. In the presence of the barium, red spots with
an RF value of about 0·10 appear on the chromatogram.
C. DETERMINATION OF HYDROGEN PEROXIDE
1. PRINCIPLE
The iodometric determination of hydrogen peroxide is based on the
following reaction:
H
2O2+2 H++2 I−→I2−2 H2O
This conversion proceeds slowly but can be accelerated by the addition
of ammonium molybdate. The iodine formed is determined titrimetri-
cally against sodium thiosulphate and is a measure of the hydrogen
peroxide content.
2. DEFINITION
The hydrogen peroxide content measured in the manner described
below is expressed as a percentage by mass (% m/m) of the product.
3. REAGENTS
All reagents should be of analytical purity.
3.1. 2N sulphuric acid3.2. Potassium iodide
3.3. Ammonium molybdate
3.4. 0·1N sodium thiosulphate3.5. 10 % (m/v) potassium iodide solution, to be freshly prepared immedi-
ately before use
3.6. 20 % (m/v) ammonium molybdate solution3.7. 1 % (m/v) starchsolution
4. APPARATUS AND EQUIPMENT
4.1. Beakers, 100 ml
4.2. Burette, 50 ml
4.3. Standard flasks, 250 ml4.4. Measuring cylinders, 25 and 100 ml
4.5. One-mark pipettes, 10 ml1982L0434 — EN — 10.04.1990 — 001.001 — 7
▼B
4.6. Conical flasks, 250 ml
5. METHOD
5.1. Into a 100-ml beaker weighout 10 g (m gram) of th e product,
containing about 0·6 g of hydrogen peroxide. Transfer the contents
withwater into a 250-ml standard flask, make up to th e mark with
water and mix.
5.2. Pipette 10 ml of the sample solution (5.1) into a 250-ml conical flask
(4.6) and add successively 100 ml of 2N sulphuric acid (3.1), 20 ml of
potassium iodide solution (3.5) and three drops of ammonium molyb-
date solution (3.6).
5.3. Titrate the iodine formed immediately against 0·1N sodium thiosulphate
solution (3.4) and just before the end point is reached add a few milli-
litres of starchsolution as indicator (3.7). Record th e consumption of
0·1N sodium thiosulphate (3.4) in millilitres (V).
5.4. In the manner described in Sections 5.2 and 5.3, carry out a blank
determination, replacing the 10 ml of the sample solution by 10 ml of
water. Record the consumption of 0·1N sodium thiosulphate in the
blank determination (Vo ml).
6. CALCULATION
Calculate the hydrogen peroxide content of the product as a percentage
by mass (% m/m) with the aid of the following formula:
% hydrogen peroxide ¼V/C0Vo ðÞ /C2 1/C17008 /C2250/C2100
m/C210/C21000
¼V/C0Vo ðÞ /C2 4/C1252
m
in which:
m = the quantity in grams of product analyzed (5.1),
Vo = the consumption in millilitres of 0·1N thiosulphate solution in
the blank determination (5.4),
V = the consumption in millilitres of 0·1N thiosulphate solution in
the titration of the sample solution (5.3).
7. REPEATABILITY(1)
For a product containing about 6 % m/m hydrogen peroxide the differ-
ence between the results of two determinations in parallel carried out on
the same sample should not exceed an absolute value of 0·2 %.
II.IDENTIFICATION AND SEMI-QUANTITATIVE DETERMINATION
OF CERTAIN OXIDATION COLORANTS IN HAIR DYES
1. PURPOSE AND SCOPE
This method is suitable for the identification and semi-quantitative
determination of the following substances in hair dyes in cream or
liquid form:
Substances Symbol
Phenylenediamines
o-Phenylenediamine (OPD)
m-Phenylenediamine (MPD)
p-Phenylenediamine (Annex V) (PPD)1982L0434 — EN — 10.04.1990 — 001.001 — 8
(1) See Norm ISO 5725.
▼B
Substances Symbol
Methylphenylenediamines
4-Methyl-1,2-phenylenediamine (toluene-3,4-
diamine)(OTD)
4-Methyl-1,3-phenylenediamine (toluene-2,4-
diamine)(MTD)
2-Methyl-1,4-phenylenediamine (toluene-2,5-
diamine)(PTD)
Diaminophenols
2,4-diaminophenol (DAP)
Hydroquinone
1,4 Benzenediol (H)
α-Naphthol (α-N)
Pyrogallol
1,2,3-trihydroxybenzene (P)
Resorcinol
1,3-dihydroxybenzene (R)
2. PRINCIPLE
Oxidation colorants are extracted at pH 10 with96 % eth anol from dyes
in cream or liquid form and identified by thin-layer chromatography,
either one- or two-dimensional.
For semi-quantitative determination of these substances, the chromato-
gram of the samples is compared by means of four developing systems
with those for reference substances produced at the same time and
under as similar conditions as possible.
3. REAGENTS
All reagents should be of analytical purity.
3.1. Ethanol, anhydrous3.2. Acetone
3.3. Ethanol, 96 % v/v
3.4. Ammonia solution, 25 %
(d
420= 0·91)
3.5. L(+)-ascorbic acid3.6. Chloroform3.7. Cyclohexane
3.8. Nitrogen, technical grade
3.9. Toluene3.10. Benzene3.11. n-Butanol3.12. Butan-2-ol
3.13. Hypophosphorous acid, 50 % v/v solution
3.14. Diazo reagent. Either:
— 3-Nitro-1-benzenediazonium chlorobenzenesulphonate, (stabilized
salt form) as in Red 2 JN — Francolor,
— 2-Chloro-4-nitro-1-benzenediazonium naphthalenebenzoate (stabi-
lized salt form) as in NNCD Reagent — reference No 74150
FLUKA,
or an equivalent.1982L0434 — EN — 10.04.1990 — 001.001 — 9
▼B
3.15. Silver nitrate
3.16. p-Dimethylaminobenzaldehyde
3.17. 2,5-Dimethylphenol3.18. Ferric chloride hexahydrate3.19. Hydrochloric acid, 10 % m/v solution
3.20. Reference substances
The reference substances are those shown in paragraph 1 ‘Purpose and
scope’. In the case of amine compounds, the reference substance must
be either the hydrochloride (mono or di) or the free base.
3.21. Reference solutions 0·5 % (m/v)
A 0·5 % (m/v) solution of eachof th e reference substances in Section
3.20 is prepared.
Weighout 50 mg ± 1 mg of h e reference substance in a standard 10-ml
flask.
Add 5 ml of 96 % ethanol (3.3) and 250 mg of ascorbic acid (3.5).Make the solution alkaline by addition of the ammonia solution (3.4) to
give an apparent pH of 10 (test withindicator paper).
Make up to 10 ml with96 % eth anol (3.3) and mix.The solutions may be kept for a week in a cool place away from the
light.
In certain cases, after the addition of the ascorbic acid and the
ammonia, a precipitate may form. It should then be allowed to settle
before proceeding.
3.22. Developing solvents
3.22.1. Acetone — chloroform — toluene (35: 25: 40 by vol)3.22.2. Chloroform — cyclohexane — absolute ethanol — 25 % ammonia (80:
10: 10: 1 by vol)
3.22.3. Benzene — butan-2-ol — water (50: 25: 25 by vol). Shake well and use
the upper phase after separating at room temperature (20 to 25 ºC).
3.22.4. n-Butanol — chloroform — reagent M (7: 70: 23 by vol). Separate
carefully at room temperature (20 to 25 ºC) and use the lower phase.
Preparation of reagent M
Ammonia solution, 25 % (v/v) 24 volumes
Hypophosphorous acid, 50 % (3·13) 1 volume
Water 75 volumes
Note
Developing solvents containing ammonia must be well shaken immedi-
ately before use.
3.23. Indicator sprays
3.23.1.Diazo reagent
Make a 5 % (m/v) aqueous solution of the chosen reagent (3.14). This
solution must be freshly prepared just before use.
3.23.2.Ehrlich's reagent
Dissolve 2 g of p-diamethylaminobenzaldehyde (3.16) in 100 ml of
hydrochloric acid 10 % (m/v) aqueous solution (3.19).
3.23.3.2,5-dimethylphenol - ferric chloride hexahydrate
Solution 1: Dissolve 1 g of dimethylphenol (3.17) in 100 ml of 96 %
ethanol (3.3).1982L0434 — EN — 10.04.1990 — 001.001 — 10
▼B
Solution 2: Dissolve 4 g of ferric chloride hexahydrate (3.18) in 100 ml
of 96 % ethanol (3.3).
For development, these solutions are sprayed separately, first solution 1
then solution 2.
3.23.4.Ammoniacal silver nitrate
25 % ammonia (3.4) is added to 5 % (m/v) aq. solution of silver nitrate
(3.15) until the precipitate just dissolves. This reagent must be prepared
immediately before use.
Do not keep.
4. APPARATUS
4.1. Usual laboratory equipment for thin-layer chromatography.
4.1.1. Plastics or glass cover to constructed that the chromatographic plate can
be surrounded with0nitrogen during application of the0spots and drying.
This precaution is necessary because of the susceptibility to oxidation
of certain colorants.
4.1.2. Micro-syringe, 10 µl, graduated in 0·2 µl divisions, witha square cut
needle, or, better, a 50 µl repeating dispenser, mounted on a clamp
stand in such a way that the plate can be kept under nitrogen.
4.1.3. Silica gel thin layer plates, ready to use, 0·25 mm thick, formed by
20 × 20 cm (Macherey and Nagel, Silica G-HR, which are on plastics
support, or equivalent)
4.2. Centrifuge, 4 000 rev./min.
4.3. Centrifuge tubes, 10 ml withPTFE-lined screw caps, or equivalent
5. PROCEDURE
5.1. Treatment of test samples
Discard the first 2 or 3 cm of cream extruded from the tube.
Put the following into a centrifuge tube (4.3) previously flushed out
withnitrogen: 300 mg ascorbic acid with3 g cream or 3 g h omoge-
nized liquid.
Add 25 % ammonia drop-wise (3.4) until the pH is 10. Make up to
10 ml with96 % eth anol (3.3).
Homogenize under nitrogen (3.8), stopper and then centrifuge at
4 000 rev./min. for 10 minutes.
Use the supernatant liquid.
5.2. Chromatography
5.2.1.Spotting the plates
Under an atmosphere of nitrogen (3.8), apply to a chromatography plate
(4.1.3) 1 µl of eachof th e above-described reference solutions at nine
points situated about 1·5 cm apart along a line approximately 1·5 cm
from the edge of the plate.
These reference solution spots are arranged as follows:
123456789
R P H PPD DAP PTD OPD OTD MPD
MTD α-N
In addition, apply at points 10 and 11 respectively 2 µl of the test solu-
tion samples obtained according to Section 5.1.
Keep the plate under nitrogen (3.8) until the moment when it is chro-
matographed.
5.2.2.Development
Place the plate in a tank previously flushed out with nitrogen (3.8),
saturated withone of th e four solvents (3.22) and allow to develop at1982L0434 — EN — 10.04.1990 — 001.001 — 11
▼B
room temperature (20 to 25 ºC) in the dark until the solvent front has
moved about 15 cm from the baseline.
Remove the plate and dry under nitrogen (3.8) at room temperature.
5.2.3.Spraying
Spray the plate immediately with one of the four solutions specified in
3.23.
5.2.4.Identification
Compare the Rfvalue and the colour obtained from the sample with
those of the reference substances chromatographed.
Table I gives as examples the Rfvalues and colours for eachsubstance
depending on the solvent and indicator used.
Confirmation of doubtful identification may sometimes be achieved by
a spiking method, adding the corresponding reference substance solu-
tion to the sample extract.
5.2.5.Semi-quantitative estimation
Compare visually the intensity of the spots for each substance identified
in 5.2.4 withan appropriate range of concentrations of th e reference
substances.
If the concentration of one or more of the substances found in the
sample is excessive, dilute the sample extract and repeat the measure-
ment.1982L0434 — EN — 10.04.1990 — 001.001 — 12
TABLE I
Rfvalues and colours obtained immediately after spraying
Reference
substance
(3.20)Developing solvents Indicator sprays
Rfvalues Resultant colours
(3.22.1) (3.22.2) (3.22.3) (3.22.4)Diazo
(3.23.1)Ehrlich
(3.23.2)Dimethylphenol
(3.23.3)AgNO
(3.23.4)
OPD 0·62 0·60 0·30 0·57 pale brown — — pale brown
MPD 0·40 0·60 0·47 0·48 violet-brown (*) yellow pale brown pale brown
PPD 0·20 0·50 0·30 0·48 brown bright red (*) violet grey
OTD 0·60 0·60 0·53 0·60 brown (*) pale orange pale brown greyishbrown
MTD 0·40 0·67 0·45 0·60 reddishbrown (*) yellow brown black
PTD 0·33 0·65 0·37 0·70 brown orange violet (*) grey
DAP 0·07 — 0 0·05 brown (*) orange violet brown
H 0·50 0·35 0·80 0·20 — orange violet black (*)
α-N 0·90 0·80 0·90 0·75 orange-brown — violet (*) black
P 0·37 — 0·67 0·05 brown very pale violet very pale brown brown (*)
R 0·50 0·37 0·80 0·17 orange (*) pale violet very pale brown pale brown
Note
1. OPD is only weakly shown up; the solvent (3.22.3) must be used to separate it clearly from the OTD.
2. (*) Indicates the best colour development.▼B1982L0434 — EN — 10.04.1990 — 001.001 — 13
▼B
6. EXAMINATION BY TWO-DIMENSIONAL THIN-LAYER CHRO-
MATOGRAPHY
This two-dimensional chromatographic procedure requires the use of
additional standards and reagents.
6.1. Additional reference solutions and substances
6.1.1. β-naphthol ( β-N)
6.1.2. 2-aminophenol (OAP)6.1.3. 3-aminophenol (MAP)6.1.4. 4-aminophenol (PAP)6.1.5. 2-nitro-1,4-phenylenediamine (2-NPPD)
6.1.6. 4-nitro-1,2-phenylenediamine (4-NOPD)
Prepare a 0·5 % m/v solution of eachof th e additional reference
substances as described in 3.21.
6.2. Additional developing solvent
6.2.1. Ethyl acetate — cyclonexane — ammonia solution, 25 % (65: 30: 0·5
by vol)
6.3. Additional indication system
Place a glass vessel in a developing tank for thin-layer chromatography,
add about 2 g crystallized iodine and close the tank with an adequate
lid.
6.4. Chromatography
6.4.1. Draw two lines, at shown in Figure 1, on the absorbent surface of a
thin-layer plate (4.1.3).
6.4.2. Under a nitrogen atmosphere (4.1.1), apply 1 to 4 µl extract (5.1) at
base point 1 (Figure 1) which is at 2 cm from the two sides. The quan-
tity of extract depends on the intensity of the spots on the
chromatograms 5.2.
6.4.3. Divide between points 2 and 3 (Figure 1) the oxidation colorants iden-
tified or assumed to be identified by 5.2 (distance between points
1·5 cm). Apply 2 µl of eachof th e reference solutions — except DAP
of which 6 µl must be applied. Conduct the operation under nitrogen
(6.4.2).
6.4.4. Repeat the operation in 6.4.3 at base points 4 and 5 (Figure 1) and keep
the plate under nitrogen until the moment it is chromatographed
(distance between points 1·5 cm).
6.4.5. Flush out a chromatographic tank with nitrogen (3.8) and place in it a
suitable quantity of developing solvent 3.22.2. Place the plate (6.4.4) in
the tank and develop it in the first elution direction (Figure 1) in the
dark.
Elute until the solvent front reaches the line marked on the plate
(approximately 13 cm).
6.4.6. Remove the plate from the tank and place it in the chromatography tank
previously flushed out with nitrogen to evaporate the elution solvent for
at least 60 minutes.
6.4.7. Witha graduated test-tube, place a suitable quantity of elution solvent
(6.2) in a tank flushed out with nitrogen (3.8), place the plate rotated
through 90º in the tank (6.4.6) and chromatograph in the second direc-
tion (also in the dark) until the solvent front reaches the line drawn on
the absorbent surface. Remove the plate from the tank and evaporate
the elution solvent in air.
6.4.8. Place the plate for 10 minutes in the chromatography tank with iodine
vapour (6.3) and interpret the two-way chromatogram using the R
fand
colour values of the reference substances chromatographed at the same
time (Table II gives a guide to the Rfvalues and colours).
Note
To obtain maximum colouring of the spots leave the chromatogram
exposed to the atmosphere for half an hour after developing.1982L0434 — EN — 10.04.1990 — 001.001 — 14
▼B
6.4.9. The presence of the oxidation colorants found in 6.4.8 can be defini-
tively confirmed by repeating the operations described in 6.4.1 to 6.4.8
and adding at base point 1 on top of the amount of extract specified in
6.4.2 1 µl of the reference substances identified in 6.4.8. If no other
spot is found in comparison with the chromatogram obtained in 6.4.8,
the interpretation of chromatogram 6.4.8 is correct.1982L0434 — EN — 10.04.1990 — 001.001 — 15
▼B
TABLE II
Colour of the reference substances after chromatography and developing
with iodine vapour
Reference
substancesColour after developing withiodine vapour
R beige
P brown
α-N violet
β-N pale brown
H violet-brown
MPD yellowishbrown
PPD violet-brown
MTD dark brown
PTD yellowishbrown
DAP dark brown
OAP orange
MAP yellowishbrown
PAP violet-brown
2-NPPD brown
4-NOPD orange
Figure 1
1982L0434 — EN — 10.04.1990 — 001.001 — 16
▼B
III.IDENTIFICATION AND DETERMINATION OF NITRITE
A. IDENTIFICATION
1. PURPOSE AND SCOPE
This method is suitable for the identification of nitrite in cosmetic
products, particularly creams and pastes.
2. PRINCIPLE
The presence of nitrite is indicated by the formation of coloured deriva-
tives with 2-aminobenzaldehyde phenylhydrazone (Nitrin®).
3. REAGENTS
All reagents should be of analytical purity.
3.1. Dilute sulphuric acid: dilute 1 ml concentrated sulphuric acid (d420=
1·84) with11 ml distilled water.
3.2. Dilute hydrochloric acid: dilute 1 ml concentrated hydrochloric acid
(d420= 1·19) with11 ml distilled water.
3.3. Methanol3.4. A solution of 2-aminobenzaldehyde phenylhydrazone (Nitrin ® reagent)
in methanol.
Weighout 2·0 g of Nitrin ® and transfer th is quantitatively into a 100-
ml standard flask. Add drop-wise 4 ml dilute hydrochloric acid (3.2)
and shake. Fill up to the mark with methanol and mix until the solution
is completely clear. Store the solution in a brown glass bottle (4.3).
4. APPARATUS
4.1. Beakers, 50 ml4.2. Standard flask, 100 ml4.3. Brown glass bottle, 125 ml4.4. Glass plate, 10 × 10 cm4.5. Plastics spatula
4.6. Filter paper, 10 × 10 cm
5. PROCEDURE
5.1. Spread part of the sample to be examined evenly over the glass plate
(4.4) so as to cover the surface to a thickness of no more than 1 cm.
5.2. Soak a sheet of filter paper (4.6) in distilled water. Lay it on the sample
and press the filter paper down with the plastics spatula (4.5).
5.3. Wait about one minute and apply to the centre of the filter paper:
— two drops of dilute sulphuric acid (3.1),
— followed by two drops of the Nitrin ® solution (3.4).
5.4. After five to 10 seconds, remove the filter paper and examine it against
daylight. The presence of nitrite is indicated by a reddish purple colora-
tion.
If the nitrite content is low, the reddish purple colour changes to yellow
after five to 15 seconds. This change of colour takes place only after
one to two minutes where large quantities of nitrite are present.
6. COMMENT
The intensity of the reddish purple colour and the time that elapses
before the change to yellow may give an indication of the nitrite
content of the sample.1982L0434 — EN — 10.04.1990 — 001.001 — 17
▼B
B. DETERMINATION
1. PURPOSE
The method describes the determination of nitrite in cosmetic products.
2. DEFINITION
The nitrite content of the sample, determined according to this method,
is expressed in % by mass of sodium nitrite.
3. PRINCIPLE
After diluting the sample with water and clarifying, the nitrite present is
made to react with sulphanilamide and N-1-naphthylethylenediamine
and the optical density of the colour obtained is measured at 538 nm.
4. REAGENTS
All reagents should be of analytical quality.
4.1. Clarification reagents these reagents may not be used more than one
week after preparation.
4.1.1. Carrez I reagent:
Dissolve 106 g of potassium cyanoferrate (II) K4Fe(CN)6·3H2O, in
distilled water and dilute withwater to 1000 ml.
4.1.2. Carrez II reagent:
Dissolve 219·5 g of zinc acetate, Zn(CH3COO)2·2H2O and 30 ml of
glacial acetic acid in distilled water and dilute withwater to 1 000 ml.
4.2. Sodium nitrite solution:
Dissolve 0·500 g of sodium nitrite in distilled water in a 1 000-ml volu-
metric flask and dilute with water to the mark. Dilute 10·0 ml of this
stock standard solution to 500 ml; 1 ml of the latter solution = 10
micrograms of NaNO2.
4.3. 1N sodium hydroxide solution4.4. 0·2 % sulphanilamide hydrochloride solution:
Dissolve 2·0 g of sulphanilamide in 800 ml of water by warming. Cool
and add 100 ml of concentrated hydrochloric acid while stirring. Dilute
withwater to 1 000 ml.
4.5. 5N hydrochloric acid4.6. N-1-naphthyl reagent:
This solution must be prepared on the day of use. Dissolve 0·1 g of N-
1-naphthylethylenediamine dihydrochloride in water and dilute with
water to 100 ml.
5. APPARATUS
5.1. Analytical balance5.2. Volumetric flasks of 100, 250, 500 and 1 000 ml5.3. Bulb or graduated pipettes
5.4. Measuring cylinders of 100 ml
5.5. Fluted filter papers, free of nitrite, diameter 15 cm5.6. Water-bath
5.7. Spectrophotometer with optical cells of 1 cm path-length
5.8. pH meter5.9. Microburette of 10 ml5.10. Beakers of 250 ml1982L0434 — EN — 10.04.1990 — 001.001 — 18
▼B
6. PROCEDURE
6.1. Weigh about 0·5 g (m gram) to a precision of 0·1 mg of the homoge-
nized sample, transfer withh ot distilled water quantitatively into a 250-
ml beaker (5.10) and bring the volume to approximately 150 ml with
hot distilled water. Place the beaker (5.10) in a water-bath (5.6) at
80 ºC for half an hour. During this period, shake the contents occasion-
ally.
6.2. Cool to room temperature and add successively, while stirring, 2 ml of
the Carrez I reagent (4.1.1) and 2 ml of the Carrez II reagent (4.1.2).
6.3. Add 1N sodium hydroxide solution (4.3) to bring the pH to 8·3. (Use
the pH meter (5.8)). Transfer the contents quantitatively to a 250-ml
volumetric flask (5.2) and make up to the mark with distilled water.
6.4. Mix the contents and filter through a fluted filter paper (5.5).6.5. Pipette (5.3) a suitable aliquot (V ml) of the clear filtrate, but not more
than 25 ml, into a 100-ml volumetric flask (5.2) and add distilled water
to a volume of 60 ml.
6.6. After mixing, add 10·0 ml of sulphanilamide hydrochloride solution
(4.4) and then 6·0 ml of 5N hydrochloric acid (4.5). Mix and allow to
stand for five minutes. Add 2·0 ml of N-1-naphthyl reagent (4.6), mix
and allow to stand for three minutes. Dilute with water to the mark and
mix.
6.7. Prepare a blank test by repeating the operations 6.5 and 6.6 without
addition of the N-1-naphthyl reagent (4.6).
6.8. Measure (5.7) the optical density at 538 nm of the solution obtained
under 6.6 using the blank solution (6.7) as a reference.
6.9. Read from the calibration graph (6.10) the sodium nitrite content in
micrograms per 100 ml of the solution (m
1micrograms) that corre-
sponds to the optical density measured in 6.8.
6.10. Using the 10 µg per ml sodium nitrite solution (4.2), prepare a calibra-
tion graphfor concentrations of 0, 20, 40, 60, 80, 100 µg of sodium
nitrite per 100 ml.
7. CALCULATION
Calculate the sodium nitrite content of the sample in percent by mass
with the aid of the following formula:
% NaNO 2¼250
V/C2m1/C210/C06/C2100
m¼m1
V/C2m/C240
in which:
m = the mass of the sample in grams taken for analysis (6.1),
m1= the sodium nitrite content in micrograms found in 6.9,
V = the number of millilitres of the filtrate used for the measure-
ment (6.5).
8. REPEATABILITY(1)
For a content of about 0·2 % m/m of sodium nitrite the difference
between the results of two determinations in parallel carried out on the
same sample should not exceed an absolute value of 0·005 %.
IV.IDENTIFICATION AND DETERMINATION OF FREE FORMALDE-
HYDE
1. PURPOSE AND SCOPE
This method describes the identification and two determination
according to the presence or not of formaldehyde donors. It is applic-
able to all cosmetic products.1982L0434 — EN — 10.04.1990 — 001.001 — 19
(1) See Norm ISO 5725.▼M1
▼M1
1.1. Identification
1.2. General determination by pentane-2,4-dione colorimetry
This method applies when formaldehyde is used alone or with other
preservatives that are not formaldehyde donors.
Where this is not the case, and if the result exceeds the maximum
permitted concentration, the following method of confirmation must be
used.
1.3. Determination in the presence of formaldehyde donors
In the method mentioned above (1.2), during the derivization, the
formaldehyde donors split and lead to results that are too high
(combined and polymerized formaldehyde)
It is necessary to separate the free formaldehyde by liquid chromato-
graphy.
2. DEFINITION
The free formaldehyde content of the sample determined according to
this method is expressed as percentage by mass.
3. IDENTIFICATION3.1. Principle
Free and combined formaldehyde in a sulphuric acid medium turns
Schiff's reagent pink or mauve.
3.2. Reagents
All reagents should be of analytical purity and the water has to be
demineralized.
3.2.1. Fuchsin;
3.2.2. Sodium sulphite hydrated at 7H
2O;
3.2.3. Concentrated hydrochloric acid (d = 1,19);
3.2.4. Sulphuric acid, about 1M;
3.2.5.Schiff's reagent:
100 mg of fuchsin (3.2.1) is weighed into a beaker and dissolved in
75 ml of water at 80 ºC. After cooling, add 2,5 g of sodium sulphite
(3.2.2). Make up to 100 ml.
Use within two weeks.
3.3. Procedure
3.3.1. Weigh2 g of th e sample in a 10-ml beaker.
3.3.2. Add two drops of sulphuric acid (3.2.4) and 2 ml of Schiff's reagent
(3.2.5). This reagent must be absolutely colourless when it is used.
Shake and leave to stand for five minutes.
3.3.3. If a pink or mauve tint is observed within the five minutes, the formal-
dehyde is present in excess of 0,01 % and is to be determined by thefree and combined method (4) and, if necessary, by procedure (5).
4. GENERAL DETERMINATION BY PENTANE-2,4-DIONE COLORI-
METRY
4.1. Principle
Formaldehyde reacts with pentane-2,4-dione in the presence of ammo-
nium acetate to form 3,5-diacetyl-1,4-dihydrolutidine. This is extracted
with butan-1-ol and the absorbance of the extract is measured at
410 nm.1982L0434 — EN — 10.04.1990 — 001.001 — 20
▼M1
4.2. Reagents
All reagents should be of analytical purity and the water has to be
demineralized.
4.2.1. Anhydrous ammonium, acetate;
4.2.2. Concentrated acetic acid, d20
4= 1,05;
4.2.3. Pentane-2,4-dione freshly distilled under reduced pressure 25 mm Hg
25º — it should not exhibit any absorption at 410 nm.
4.2.4. Butan-1-ol;
4.2.5. Hydrochloric acid, 1 M;
4.2.6. Hydrochloric acid, approximately 0,1 M;4.2.7. Sodium hydroxide, 1 M;4.2.8. Starch solution freshly prepared according to the European Pharmaco-
poeia (1 g/50 ml water), 2nd edition 1980, part I-VII-1-1;
4.2.9. 37 to 40 % w/v formaldehyde;
4.2.10 Standard iodine solution, 0,05 M;
4.2.11. Standard sodium thiosulphate solution, 0,1 M;
4.2.12Pentane-2,4-dione reagent:
In a 1 000 ml volumetric flask dissolve:
— 150 g ammonium acetate (4.2.1),
— 2 ml pentane-2,4-dione (4.2.3),
— 3 ml acetic acid (4.2.2).
Make up to 1000 ml withwater (pH of solution about 6,4).This reagent must be freshly prepared;
4.2.13. Reagent (4.2.12) without pentane-2,4-dione;
4.2.14Formaldehyde-standard: stock solution
Pour 5 g of formaldehyde (4.2.9) into a 1 000-ml volumetric flask and
make up to 1 000 ml withwater.
Determine the strength of this solution as follows:Remove 10,00 ml; add 25,00 ml of a standard iodine solution (4.2.10)
and 10,00 ml of sodium hydroxide solution (4.2.7).
Allow to stand for five minutes.
Acidify with11,00 ml of HCl (4.2.5) and determine th e excess iodine
with a standard sodium thiosulphate solution (4.2.11), using starch solu-
tion (4.2.8) as indicator.
1 ml of 0,05 M iodine (4.2.10) consumed is equivalent to 1,5 mg
formaldehyde;
4.2.15.Formaldehyde-standard: diluted solution
Dilute the formaldehyde stock solution successively 1/20 and then 1/
100 withwater.
1 ml of this solution contains about 1 µg of formaldehyde.Calculate the exact content.
4.3. Apparatus
4.3.1. Standard laboratory apparatus;4.3.2. Phase separation filter, Whatman 1 PS (or equivalent);
4.3.3. Centrifuge;
4.3.4. Water-bathset at 60 ºC;4.3.5. Spectrophotometer;
4.3.6. Glass cells withan optical pathof 1 cm.1982L0434 — EN — 10.04.1990 — 001.001 — 21
▼M1
4.4. Procedure
4.4.1.Sample solution
Into a 100-ml volumetric flask weighto with in 0,001 g a quantity (in g)
of the test sample corresponding to a presumed quantity of formalde-hyde of about 150 µg.
Make up to 100 ml withwater and mix (solution S).
(Check that the pH is close to 6; if not, dilute in the hydrochloric acid
solution (4.2.6).)
To a 50-ml Erlenmeyer flask add:
— 10,00 ml of the solution S,
— 5,00 ml pentane-2,4-dione reagent (4.2.12).— demineralized water to a final volume of 30 ml.
4.4.2.Reference solution
Possible interference due to background colour in the test sample is
eliminated by the use of this reference solution:
To a 50-ml Erlenmeyer flask add:
— 10,00 ml S solution,
— 5,00 ml reagent (4.2.13),— demineralized water to a final volume of 30 ml.
4.4.3.Blank test
To a 50-ml Erlenmeyer flask add:
— 5,0 ml pentane-2,4-dione reagent (4.2.12),
— demineralized water to a final volume of 30 ml.
4.4.4.Determination
4.4.4.1. Shake the mixtures from 4.4.1, 4.4.2 and 4.4.3. Immerse the Erlenmeyer
flasks in a water-bathat 60 ºC for exactly 10 minutes. Allow to cool for
two minutes in a bathof iced water.
4.4.4.2 Transfer into 50-ml separating funnels containing 10 ml of butan-1-ol
(4.2.4). Rinse each flask with 3 to 5 ml of water. Shake the mixture
vigorously for exactly 30 seconds. Allow it to separate.
4.4.4.3. Filter the butan-1-ol phase into the measurement cells (4.3.2) through a
phase-separation filter. Centrifuging (3 000 g
nfor five minutes) may
also be used.
4.4.4.4. Measure the absorbance A1at 410 nm of the extract of the sample solu-
tion from 4.4.1 against the extract of the reference solution 4.4.2.
4.4.4.5. Similarly measure the absorbance A2of the extract of the blank solution
from 4.4.3 against butan-1-ol.
NB :All these operations must be carried out within 25 minutes from
the moment when the Erlenmeyer flasks are placed in the water
bathat 60 ºC.
4.4.5.Calibration curve
4.4.5.1. Into a 50-ml Erlenmeyer flask place:
— 5,00 ml of the diluted standard solution from 4.2.15,
— 5,00 ml of the pentane-2,4-dione reagent (4.2.12),— demineralized water to a final volume of 30 ml.
4.4.5.2. Continue as described in 4.4.4 and measure the absorbance against
butan-1-ol (4.2.4).
4.4.5.3. Repeat the procedure with 10, 15, 20 and 25 ml of the diluted standard
solution (4.2.15).
4.4.5.4. To obtain the zero value (corresponding to the coloration of the
reagents) proceed as in 4.4.4.5.
4.4.5.5. Construct the calibration curve after subtraction of the zero value from
eachof th e absorbances obtained in 4.4.5.1 and 4.4.5.3. Beer's Law is
valid up to 30 µg formaldehyde.1982L0434 — EN — 10.04.1990 — 001.001 — 22
▼M1
4.5. Calculations
4.5.1. Subtract A2from A1and read off from the calibration curve (4.4.5.5)
the amount C, in µg, of formaldehyde in the sample solution (4.4.1).
4.5.2. Calculate the formaldehyde content of sample (% m/m) with the aid of
the following formula:
formaldehyde content in % ¼C
103/C1m
where:
m = mass of the test portion in 9.
4.6. Repeatability (1)
For a formaldehyde content of 0,2 % the difference between the results
of two determinations in parallel carried out on the same sample should
not exceed 0,005 % for determination by pentane-2,4-dione colorimetry.
If the determination of free formaldehyde leads to results greater than
the maximum concentrations provided for in Directive 76/768/EEC,
i.e.:
(a) between 0,05 % and 0,2 % in a non-labelled product;
(b) greater than 0,2 % in the product, whether or not labelled
the procedure described in 5 below must be applied.
5. DETERMINATION IN THE PRESENCE OF FORMALDEHYDE
DONORS
5.1. Principle
The separate formaldehyde is transformed into a yellow lutidinic deri-
vative by a reaction withth e pentane-2,4-dione in a post-column reactor
and the derivative obtained is detected by absorbance at 420 nm.
5.2. Reagents
All reagents should be of analytical purity and the water has to be
demineralized.
5.2.1. HPLC grade water or water of equivalent quality;5.2.2. Anhydrous ammonium, acetate;5.2.3. Concentrated acetic acid;
5.2.4. Pentane-2,4-dione (kept at 4 ºC);
5.2.5. Anhydrous disodium phosphate;5.2.6. 85 % orthophosphoric acid (d = 1,7);5.2.7. HPLC grade methanol;5.2.8. Dichloromethane;
5.2.9. 37 to 40 % w/v formaldehyde;
5.2.10. Sodium hydroxide, 1 M;5.2.11. Hydrochloric acid, 1 M;5.2.12. Hydrochloric acid, 0,002 M;5.2.13. Starch solution freshly prepared according to the European Pharmaco-
poeia (see 4.2.8);
5.2.14. Standard iodine solution, 0,05 M;5.2.15. Standard sodium thiosulphate solution, 0,1 M;
5.2.16.Mobile phase:
Aqueous solution of disodium phosphate (5.2.5), 0,006 M adjusted to
pH 2,1 with orthophosphoric acid (5.2.6);1982L0434 — EN — 10.04.1990 — 001.001 — 23
(1) ISO 5725.
▼M1
5.2.17.Post-column reagent:
In a 1 000 ml volumetric flask dissolve:
— 62,5 g ammonium acetate (5.2.2),
— 7,5 ml acetic acid (5.2.3),
— 5 ml pentane-2,4-dione (5.2.4).
Make up to 1000 ml withwater (5.2.1).Keep this reagent away from the light.Conservation time: maximum three days at 25 ºC.No change in colour should be observed;
5.2.18.Formaldehyde standard: stock solution
Pour 10 g of formaldehyde (5.2.9) into a 1 000 ml volumetric flask and
make up to 1 000 ml withwater.
Determine the strength of this solution as follows:Remove 5,00 ml; add 25,00 ml of the standard iodine solution (5.2.14)
and 10,00 ml of the sodium hydroxide solution (5.2.10).
Allow to stand for five minutes.Acidify with11,00 ml of HCl (5.2.11) and titrate th e excess standard
iodine solution with standard sodium thiosulphate solution (5.2.15),
using starchsolution (5.2.13) as indicator.
1 ml of iodine solution (5.2.14) is equivalent to 1,5 mg formaldehyde;
5.2.19.Formaldehyde standard: diluted solution
Dilute the stock solution to 1/100th of its initial strength in the mobile
phase (5.2.16).
1 ml of this solution contrains about 37 mg formaldehyde.Calculate the exact content.
5.3. Apparatus
5.3.1. Standard laboratory apparatus;5.3.2. HPLC pump, pulsation-free;5.3.3. Low-pressure pulsation-free pump for the reagent (or a second HPLC
pump);
5.3.4. Injection valve witha 10 µl loop;5.3.5. Post-column reactor withth e following components:
+ one 1-litre three-neck flask,
+ one 1-litre flask heater,
+ two Vigreux columns witha minimum of 10 plates, two air-cooled,+ stainless steel tube (for heat exchange) 1,6 mm — internal diameter
0,23 mm, length= 400 mm,
+ Teflon tube 1,6 mm — internal diameter 0,30 mm, length5 m
(Frenchknitting) see Appendix 1),
+ one T-piece without any dead volume (Valco or equivalent),+ three unions without any dead volume
Or:one post-column module Applied Biosystems PCRS 520 or equiva-
lent fitted witha 1-ml reactor;
5.3.6. Membrane filter, pore size 0,45 µm;5.3.7. SEP-PAK
RC18cartridge or equivalent;
5.3.8.Ready-to-use colulmns:
— Bischoff hypersil RP 18 (type NC reference C 25.46 1805)
(5 µm, length= 250 mm, internal diameter = 4,6 mm),
— or Dupont, Zorbax ODS
(5 µm, length= 250 mm, internal diameter = 4,6 mm),
— or Phase SEP, spherisorb ODS 2
(5µm, length= 250 mm, internal diameter = 4,6 mm);1982L0434 — EN — 10.04.1990 — 001.001 — 24
▼M1
5.3.9Pre-column
Bischoff K1hypersil RP 18 (reference K1 G 6301 1805)
(5µm, length= 10 mm, or equivalent).
5.3.10 The column and pre-column are connected by means of an Ecotube
system (reference A 15020508 Bischoff) or equivalent.
5.3.11. Assemble the apparatus (5.3.5) as shown in the block diagram in
Appendix 2.
The connections after the injection value must be kept as short as
possible. In this case, the stainless-steel tube between the reactor outlet
and the detector inlet is intended to cool the mixture prior to detection
and the temperature in the detector is unknown but constant;
5.3.12. UV visible detector;
5.3.13. Recorder;
5.3.14. Centrifuge;5.3.15 Ultrasonic bath;5.3.16. Vibrating stirrer (vortex or equivalent).
5.4. Procedure
5.4.1.Calibration curve
This is produced by plotting peak heights as a function of the concen-
tration of formaldehyde standard: diluted.
Prepare the standard solutions by diluting the formaldehyde reference
solution (5.2.19) with the mobile phase (5.2.16):
— 1,00 ml of solution (5.2.19) diluted to 20,00 ml (about 185 µg/
100 ml)
— 2,00 ml of solution (5.2.19) diluted to 20,00 ml (about 370 µg/
100 ml)
— 5,00 ml of solution (5.2.19) diluted to 25,00 ml (about 740 µg/
100 ml)
— 5,00 ml of solution (5.2.19) diluted to 20,00 ml (about 925 µg/
100 ml)
The standard solutions are kept for one hour at laboratory temperature
and must be freshly prepared.
The linearity of the calibration curve is good for concentrations
between 1,00 and 15,00 µg/ml.
5.4.2.Preparation of the samples
5.4.2.1. Emulsions (creams, make-up base, eyeliners)
Into a stoppered 100-ml flask weighto th e nearest 0,001 g a quantity of
test sample (m g) corresponding to a presumed quantity of 100 µg of
formaldehyde. Add 20,00 ml dichloromethane (5.2.8) and 20,00 ml
hydrochloric acid (5.2.12), accurately measured. Mix with the vibrating
stirrer (5.3.16) and by means of the ultrasonic bath (5.3.15). Separate
the two phases by centrifuging (3 000 g
nfor two minutes). Meanwhile,
wash a cartridge (5.3.7) with 2 ml methanol (5.2.7), then condition with
5 ml water (5.2.1).
Pass 4 ml of the aqueous phase of the extract through the conditioned
cartridge, discard the first 2 ml and recover the following fraction.
5.4.2.2. Lotions, shampoos
Weighinto a stoppered 100-ml flask to th e nearest 0,001 g a quantity of
test sample (m g) corresponding to a presumed quantity of about 500 µg
of formaldehyde.
Make up to 100 ml with the mobile phase (5.2.16).Filter the solution through a filter (5.3.6) and inject or pass it through a
cartridge (5.3.7) conditioned as before (5.4.2.1). All the solutions must
be injected immediately after preparation.1982L0434 — EN — 10.04.1990 — 001.001 — 25
▼M1
5.4.3.Chromatographic conditions
— Flowrate of the mobile phase: 1 ml/min,
— Reagent flowrate: 0,5 ml/min,— Total flowrate at the detector outlet: 1,5 ml/min,— Injected volume: 10 µl,— Elution temperature: In the case of difficult separations, immerse
the column in a bath of melting ice: wait for the temperature to
stabilize (15-20 min),
— Temperature of post-column reaction: 100 ºC,— Detection: 420 nm.
NB:The entire chromatographic system and post-column must be
flushed out with water after use (5.2.1). Where the system is not
used for more than two days, this flushing must be followed by
flushing with methanol (5.2.7). Before reconditioning the system
pass water through it to avoid recrystallization.
5.5. Calculation
Emulsions: (5.4.2.1):
Formaldehyde content in % (m/m):
C/C110/C06/C1100
5m¼C/C110/C04
5m
Lotions, shampoos:
In this case the formula becomes:
C/C110/C06/C1100
m¼C/C110/C04
m
where:
m = mass of the sample analysed in g (5.4.2.1),
C = formaldehyde concentration in µg/100 ml read off from the cali-
bration curve (5.4.1).
5.6. Repeatability (1)
For a content of 0,05 % of formaldehyde the difference between the
results of two determinations in parallel carried out on the same sample
should not exceed 0,001 %.
For a content of 0,2 % of formaldehyde the difference between the
results of two determinations in parallel carried out on the same sample
should not exceed 0,005 %.
V.DETERMINATION OF RESORCINOL IN SHAMPOOS AND HAIR
LOTIONS
1. PURPOSE AND SCOPE
This method specifies the gas chromatographic determination of resor-
cinol in shampoos and hair lotions. The method is suitable for
concentrations of 0·1 up to 2·0 % by mass in the sample.
2. DEFINITION
The content of resorcinol in the sample as determined by this method is
expressed as percentage by mass.
3. PRINCIPLE
Resorcinol and 3,5-dihydroxytoluene, (5-methylresorcinol) added as an
internal standard, are separated from the sample by thin-layer chroma-
tography. Both compounds are isolated by scraping their spots from the1982L0434 — EN — 10.04.1990 — 001.001 — 26
(1) ISO 5725.▼B
▼B
thin-layer plate and extracting with methanol. Finally the extracted
compounds are dried silylated and determined by gas chromatography.
4. REAGENTS
All reagents must be of analytical grade.
4.1. Hydrochloric acid 25 % (m/m)4.2. Methanol4.3. Ethanol 96 % (v/v)4.4. Ready made silica gel TLC sheets (plastic or aluminium) with fluores-
cent indicator. Deactivate as follows: spray ordinary pre-coated silica
sheets with water until glazed. Allow the sprayed plates to dry in air
at room temperature for one to three hours.
Note
If plates are not deactivated losses of resorcinol can occur by irrever-
sible adsorption on silica.
4.5. Developing solvent; acetone — chloroform — acetic acid (20:75:5 by
volume).
4.6. Resorcinol standard solution; dissolve 400 mg resorcinol in 100 ml of
96 % ethanol (4.3) (1 ml corresponds to 4 000 µg resorcinol).
4.7. Internal standard solution; dissolve 400 mg 3,5-dihydroxytoluene
(DHT) in 100 ml of 96 % ethanol (4.3) (1 ml corresponds to 4 000 µg
DHT).
4.8. Standard mixture; mix 10 ml of solution 4.6 and 10 ml of solution 4.7
in a 100-ml volumetric flask, make up to the mark with 96 % ethanol
(4.3) and mix (1 ml corresponds to 400 µg resorcinol and 400 µg DHT).
4.9 Silylating agents:4.9.1. N, O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA)4.9.2. Hexamethyldisilazane (HMDS)4.9.3. Trimethylchlorosilane (TMCS)
5. APPARATUS
5.1. Usual thin-layer and gas chromatography equipment5.2. Glassware
6. PROCEDURE6.1. Preparation of the sample
6.1.1. Weighaccurately into a 150-ml beaker a test portion (m grams) of th e
product which contains approximately 20 to 50 mg resorcinol.
6.1.2. Acidify with hydrochloric acid (4.1) until the mixture is acid (approxi-
mately 2 to 4 ml are needed), add 10 ml (40 mg DHT) of the internal
standard solution (4.7) and mix. Transfer to a 100-ml volumetric flask
with ethanol (4.3) make up to the mark with ethanol and mix.
6.1.3. Apply 250 µl of the solution (6.1.2) to a deactivated silica sheet (4.4) as
a continuous line of approximately 8 cm length. Take care to get the
line as narrow as possible.
6.1.4. Apply 250 µl of the standard mixture (4.8) to the same plate in the
same way (6.1.3).
6.1.5. Spot on two points of the starting line 5 µl of each of the solutions 4.6
and 4.7 to aid localizing after plate development.
6.1.6. Develop the plate in an unlined (unsaturated) tank filled with devel-
oping solvent 4.5 until the solvent front has reached a line 12 cm from
the starting line; usually this takes about 45 minutes. Air-dry the plate
and localize the resorcinol/DHT-zone under short wave UV-light
(254 nm). The two compounds have approximately the same R
fvalues.
Mark the bands with a pencil at 2 mm distance from the outside dark
borderline of the bands. Remove these zones and collect the adsorbent
of eachband in a 10-ml bottle.
6.1.7. Extract the adsorbent containing the sample and the absorbent
containing the standard mixture each in the following way:1982L0434 — EN — 10.04.1990 — 001.001 — 27
▼B
add 2 ml methanol (4.2) and extract for one hour with continuous stir-
ring. Filter the mixture and repeat the extraction for another 15 minutes
with2 ml meth anol.
6.1.8. Combine the extracts and evaporate the solvent by drying overnight in a
vacuum desiccator filled witha suitable desiccant. Do not apply any
heat.
6.1.9. Silylate the residues (6.1.8) either as indicated under 6.1.9.1 or 6.1.9.2.6.1.9.1. Add 200 µl BSTFA (4.9.1) witha microsyringe and leave th e mixture
in a closed vessel for 12 hours at room temperature.
6.1.9.2. Add successively 200 µl HMDS (4.9.2) and 100 µl TMCS (4.9.3) witha
microsyringe and heat the mixture for 30 minutes at 60 ºC in a closed
vessel. Cool the mixture.
6.2. Gas chromatography
6.2.1.Chromatographic conditions
The column must yield a resolution, R, equal to or better than 1·5,
where:
R¼
2d0r2/C0r1 ðÞ
w1þw2
in which:
r1and r2= retention times in minutes of two peaks,
w1and w2= the same peaks widths at half height in mm,
d′ = the shart speed in mm per minute.
The following column and gas chromatographic conditions have been
found suitable:
Column material: stainless steel
length: 200 cm
internal diameter: =3m m
filling: 10 % OV-17 on Chromo-
sorb WAW 100 to
120 mesh
Flame ionization detector
Temperatures:
column: 185 ºC (isothermal)
detector: 250 ºC
injection port: 250 ºC
Carrier gas: nitrogen
flow: 45 ml/min.
For settings of hydrogen and air flow follow the manufacturer's instruc-
tions.
6.2.2. Inject 1 to 3 µl of the solutions obtained under 6.1.9 in the gas chroma-
tograph. Carry out five injections for each solution (6.1.9), measure the
peak areas, average these and calculate the peak area ratio: S = peak
area resorcinol/peak area DHT.
7. CALCULATION
The concentration of resorcinol in the sample, expressed as % by mass
(% m/m), is given by:
% resorcinol ¼4
M/C2Ssample
Sstandard mixture1982L0434 — EN — 10.04.1990 — 001.001 — 28
▼B
in which:
M = test portion in grams (6.1.1),
Ssample= the average peak area ratio according to 6.2.2 for
the sample solution,
Sstandard mixture= the average peak area ratio according to 6.2.2 for
the standard mixture.
8. REPEATABILITY(1)
For a resorcinol content of about 0·5 % the difference between the
results of two determinations in parallel carried out on the same sample
should not exceed an absolute value of 0·025 %.
VI.DETERMINATION OF METHANOL IN RELATION TO ETHANOL
OR PROPAN-2-OL
1. PURPOSE AND SCOPE
This method describes the gas chromatographic analysis of methanol in
all kinds of cosmetic products (including aerosols).
Relative levels of 0 to 10 % can be determined.
2. DEFINITION
The methanol content determined according to this method is expressed
in % by mass of methanol in relation to ethanol or propan-2-ol.
3. PRINCIPLE
The determination is carried out by gas chromatography.
4. REAGENTS
Use analytical grade reagents.
4.1. Methanol4.2. Ethanol absolute4.3. Propan-2-ol4.4. Chloroform, freed from alcohols by washing with water
5. APPARATUS
5.1. Gas chromatograph:
withKath arometer detector for aerosol samples,
withflame ionization detector for non-aerosol samples.
5.2. Volumetric flasks, 100 ml5.3. Pipettes, 2 ml, 20 ml, 0 to 1 ml5.4. Microsyringes 0 to 100 µl and 0 to 5 µl
and (only for aerosol samples) special gas-tight syringe with sliding
valve, (see sampling procedure Figure 5(
2).
6. PROCEDURE6.1. Sample preparation
6.1.1. Aerosol products are sampled according to Chapter II of the Annex to
Commission Directive 80/1335/EEC of 22 December 1980(
2) and then
analyzed by gas chromatography under the conditions of 6.2.1.
6.1.2. Non-aerosol products sampled according to the abovementioned
Chapter II are diluted with water to a level of 1 to 2 % ethanol or
propan-2-ol, and then analyzed by gas chromatography under the condi-
tions of 6.2.2.1982L0434 — EN — 10.04.1990 — 001.001 — 29
(1) See Norm ISO 5725.
(2) OJ No L 383, 31. 12. 1980, p. 27.
▼B
6.2. Gas chromatography
6.2.1. For aerosol samples, the katharometer detector is used.
6.2.1.1. The column is filled with 10 % Hallcomid M18 on Chromosorb WAW
100 to 200 mesh.
6.2.1.2. The column must yield a resolution, R, equal to or better than 1·5,
where:
R¼2d0r2/C0d0r1
w1þw2
in which:
r1and r2= retention times in minutes of two peaks,
w1and w2= the same peaks widths at half height in mm,
d′ = the chart speed in mm per minute.
6.2.1.3. The following conditions allow this resolution to be achieved:
Column material: stainless steel
length: 3·5 metresdiameter: 3 mm
Katharometer bridge current: 150 mA
Carrier gas: Heliumpressure: 2·5 bar
flow: 45 ml/min.
Temperatures:
injection port: 150 ºCdetector: 150 ºC
column oven: 65 ºC
Peak area measurements can be improved by electronic integration.
6.2.2. For non-aerosol samples:
6.2.2.1. The column is filled with Chromosorb 105 or Porapak QS and the
flame ionization detector is used.
6.2.2.2. The column must yield a resolution, R, equal to or better than 1·5,
where:
R¼2
d0r2/C0d0r1
w1þw2
r1and r2= retention times in minutes of two peaks,
w1and w2= the same peaks widths at half height in mm,
d′ = the chart speed in mm per minute.
6.2.2.3. This resolution has been achieved using the following conditions:
Column material: stainless steel
length: 2 metresdiameter: 3 mm
Electrometer sensitivity: 8 × 10
−10A
Gases:
carrier: nitrogenpressure: 2·1 bar
flow: 40 ml/min.
Auxiliary gas: hydrogen1982L0434 — EN — 10.04.1990 — 001.001 — 30
▼B
pressure: 1·5 bar
flow: 20 ml/min.
Temperatures:
injection port: 150 ºCdetector: 230 ºCcolumn oven 120 to 130 ºC
7. STANDARD GRAPH
7.1. For the gas chromatography procedure 6.2.1 (Hallcomid M18 column)
use the following standard mixtures. Prepare these mixtures by
measuring withpipettes, but find th e exact amount by immediate
weighing of the pipette or flask after each addition.
Relative strength
(m/m %)Methanol
(ml)Ethanol
or
propan-2-
ol
(ml)Chloroform added to
a volume of
approximately 2 · 5 % 0 ·5 20 100 ml
approximately 5 · 0 % 1 ·0 20 100 ml
approximately 7 · 5 % 1 ·5 20 100 ml
approximately 10 · 0 % 2 ·0 20 100 ml
Inject 2 to 3 µl into the chromatograph using conditions of 6.2.1.
Calculate peak areas ratio (methanol/ethanol) or (methanol/propan-2-ol)
of eachmixture. Plot standard graphusing:
X-axis: % methanol in relation to ethanol or propan-2-ol,
Y-axis: peak area ratio (methanol/ethanol) or (methanol/propan-2-ol).
7.2. For the gas chromatography procedure 6.2.2 (Porapak QS or Chromo-
sorb 105) use the following standard mixtures. Prepare these mixtures
by measuring withmicrosyringe and pipette, but find th e exact amount
by immediate weighing of the pipette or flask after each addition.
Relative strength
(m/m %)Methanol
(µl)Ethanol
or
propan-2-
ol
(ml)Water added to a
volume of
approximately 2 · 5 % 50 2 100 ml
approximately 5 · 0 % 100 2 100 ml
approximately 7 · 5 % 150 2 100 ml
approximately 10 · 0 % 200 2 100 ml
Inject 2 to 3 µl into the chromatograph using conditions of 6.2.2.
Calculate peak area ratio (methanol/ethanol) or (methanol/propan-2-ol)
of eachmixture. Plot standard graphusing:
X-axis: % methanol in relation to ethanol or propan-2-ol,
Y-axis: peak area ratio (methanol/ethanol) or (methanol/propan-2-ol).
7.3. The standard graph must be a straight line.1982L0434 — EN — 10.04.1990 — 001.001 — 31
▼B
8. REPEATABILITY(1)
For a methanol content of 5 % relative to ethanol or propan-2-ol, the
difference between the results of two determinations in parallel carried
out on the same sample should not exceed 0·25 %.1982L0434 — EN — 10.04.1990 — 001.001 — 32
(1) See Norm ISO 5725.
▼M1
Appendix 1
INTRUCTION FOR ‘FRENCH KNITTING’
ACCESSORIES REQUIRED
— One wooden bobbin:
external diameter 5 cm with a hole of 1,5 cm diameter made through the
centre. Insert four steel nails (as shown in Figures 1 and 2). The distance
between two nails must be 1,8 cm and they must be 0,5 cm from the hole,
— one rigid needle (of the crotchet-hook type) to loop the Teflon tube,— 5 m of 1,6 mm Teflon tube, internal diameter 0,3 mm.
PROCEDURE
To start off the ‘French knitting’, the Teflon tube must be threaded from the top
of the bobbin to the bottom via the central hole (leaving around 10 cm of tube
protruding from the bottom of the bobbin, enabling the chain to be pulled
through during the knitting process); then wind the tube around the four nails
in turn as shown in Figure 3.
The top and bottom of the French knitting will be protected by metal rings and
compression screws; take care not to crush the Teflon when pulling tight. Wind
the tube around each nail for a second turn and make the ‘stitches’ as follows:
— lift the lower tube over the upper tube with the hook (see Figure 4). Repeat
this process on each of the nails in order (1, 2, 3, 4 in an anti-clockwise
direction), until 5 m or the desired length of knitting is produced.
Leave around 10 cm of tube to close the chain. Thread the tube through each of
the four loops and pull gently, to close up the end of the chain.
NB:Frenchknitting manufactured for post column reactors is available on th e
market (Supelco).1982L0434 — EN — 10.04.1990 — 001.001 — 33
▼M1
Schematic diagram of the bobbin
1982L0434 — EN — 10.04.1990 — 001.001 — 34
▼M1
Appendix 2
1 = HPLC pump
2 = Injection valve
3 = Column withpre-column4 = Reagent pump
5 = T-piece without dead volume
5′= T-piece (Vortex)
6-6′= Union without dead volume
7 = ‘Frenchknitting’
7′= Reactor
8 = Three-neck flask with boiling water9 = Flask heater
10 = Coolant
11 = Stainless steel heat-exchanger tube11′= Heat exchanger
12 = Visible UV detector
13 = PCRS 520 post-column module
5.3.5
5.3.6
1982L0434 — EN — 10.04.1990 — 001.001 — 35