legislación cosmética eurepean comission analitica

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The rules governing cosmetic productsin the European Union

Volume 2

Cosmetics legislationCosmetic products

Methods of analysis

1999 Edition

EUROPEAN COMMISSION

Enterprise Directorate-GeneralPharmaceuticals and cosmetics


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THE RULES GOVERNING COSMETIC PRODUCTS

IN THE EUROPEAN UNION

Volume 1 Cosmet ics legisla t ion

Cosmetic products

Volume 2 Methods of analys is

Cosmetic products

Volu m e 3 Gu ide lin es

Cosmetic products


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iii

FOREWORD

In t he ea rly 1970s, the Member Sta tes of the EU decided to harm onise th eir na tional cosmetic

regulat ions in order t o enable the free circulation of cosmet ic products within th e Comm un ity. As

a result of numerous discussions between experts from all Member States, Council Directive

76/768/EEC was adopted on 27 July 1976. The principles laid down in the Cosmetics Directive

take into account the needs of the consumer while encouraging commercial exchange and

eliminating barriers to trade. For example, if a product is to move freely within the EU, the same

labelling, packaging and safety regulations must apply. This is one of the main objectives of the

Cosmetics Directive: to give clear guidance on what requirements a safe cosmetic product should

fulfil in order to freely circulate within the EU, without pre-market authorisation. The Cosmetics

Directive aims to guarantee the safety of cosmetic products for human use. This safety relates to

composition, packaging and information and it falls totally under the responsibility of theproducer or t he importer into the E U who is responsible for the ma rketing liability. There is no

pre-market control for cosmetic products at Member State or EU level. Control of cosmetic

products within th e EU is assu red th rough th e responsibility of th e person who places the product

on the market , a simple notif icat ion of manufacturing/import ing si te, and an in-market

sur veillance system .

Volume 2 of the series entitled The Rules governing cosmetic products in the European Union

incorporates the seven Commission Directives on the approximation of the laws of the Member

States relating to methods of analysis necessary for checking the composition of cosmetic

products.

Directive 76/768/EEC provides for the official testing of cosmetic products with the aim ofensuring that the condit ions prescribed pursuant to Community provisions concerning the

composition of the cosmetic products are satisfied. Effective in-market control by Member States

ensures that only cosmetic products which conform to the provisions of the Cosmetics Directive

and its Annexes are on the market. Inspectors appointed at national level may visit department

stores, supermarkets, small shops and market stal ls to check the products being sold. If

necessary, these inspectors may take any product from the market to official laboratories to be

tested for compliance with EU regulations. Article 8(1) of Directive 76/768/EEC provides for the

determination of the methods of analysis necessary for checking the composition of cosmetic

products. A certain number of methods of analysis have already been validated at European level

and accepted as official methods and described in seven Commission Direct ives on the

approximation of the laws of the Member States relating to methods of analysis necessary for

checking the composition of cosmetic products. This means that official testing of cosmetic

products by laboratories of any kind (national, control, etc.) have to be carried out in accordance

with t he E ur opean official met hods described in t hese Directives.

Commission Direct ives 80/1335/EEC and 82/434/EEC have already been amended once

respectively by Commission Directive 87/143/EEC and Commission Directive 90/207/EEC. With a

view to facilitating consultation, there are set out here in codified form for internal use by the

competent Commission departments. These codified texts are available to the public but have no

force in law. Where doubts exist, the original texts as published in the Official Journal of the

Eu ropean Commun ities, should be consu lted.


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TABLE OF CONTENTS

FOREWORD .......................................................................................................................... III

FIRST COMMISSION DIRECTIVE 80/1335/EEC......................................................... 1Firs t Comm ission Dir ective 80/1335/EE C of 22 December 1980 on t he a pproxima tion of th e

laws of the Member States relating to methods of analysis necessary for checking the

comp osit ion of cosm et ic pr odu cts ................................................................................................................... 1

SECOND COMMISSION DIRECTIVE 82/434/EEC ..................................................... 27Second Commission Directive 82/434/EEC of 14 May 1982 on the approximation of the laws

of the Member States relat ing to methods of analysis necessary for checking the

comp osit ion of cosmet ic pr odu cts ................................................................................................................... 27

THIRD COMMISSION DIRECTIVE 83/514/EEC .......................................................... 65Third Comm ission Directive 83/514/EE C of 27 Septem ber 1983 on t he a pproxima tion of th e


comp osit ion of cosmet ic pr odu cts ................................................................................................................... 65

FOURTH COMMISSION DIRECTIVE 85/490/EEC...................................................... 105Fourth Commission Directive 85/490/EEC of 11 October 1985 on the approximation of the


comp osit ion of cosm et ic pr odu cts ................................................................................................................... 105

FIFTH COMMISSION DIRECTIVE 93/73/EEC.............................................................. 129Fifth Commission Directive 93/73/EEC of 9 September 1993on the methods of analysis

necessaryfor checkin g composit ion of cosm et ic pr oduct s ....................................................................... 129

SIXTH COMMISSION DIRECTIVE 95/32/EC ................................................................ 157Sixth Comm ission Dir ective 95/32/EC of 7 J uly 1995 relat ing to met hods of an alysis

necessa ry for ch eckin g th e composit ion of cosmet ic products ................................................................ 157

SEVENTH COMMISSION DIRECTIVE 96/45/EC......................................................... 177Seventh Commission Directive 96/45/EC of 2 July 1996 relating to methods of analysis

necessa ry for ch eckin g th e composit ion of cosmet ic products ................................................................ 177


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FIRST COMMISSION DIRECTIVE 80/1335/EEC

First Commission Directive 80/1335/EEC of 22 December 1980

on the approximation of the laws of the Member States relating

to methods of analysis necessary for checking the composition

of cosmetic products

(As am ended by Com m ission Directive 87/ 143/ EE C of 10 February 1987)

THE COMMISSION OF THE EU ROPEAN COMMUNITIES,

Ha ving regard t o the Tr eat y establishing the Eur opean E conomic Comm unity,

Ha ving regar d t o Council Directive 76/768/EE C of 27 J uly 1976 on th e a pproxima tion of th e laws

of the Member States relating to cosmetic products(1), as amended by Directive 79/661/EEC(2),

an d in par ticular Art icle 8(1) thereof,

Wherea s Directive 76/768/EE C provides for th e official t esting of cosmet ic products with th e a im

of ensuring that the conditions prescribed pursuant to Community provisions concerning the

composition of th e cosm etic products a re s at isfied;

Whereas all the necessary meth ods of ana lysis mu st be est ablished as soon a s possible; whereas

the laying down of methods for the sampling, laboratory preparat ion, identif icat ion and

determ ination of free sodium a nd pota ssium hydroxides, the identification a nd det ermina tion of

oxalic acid and alkaline salts thereof in hair care products, the determination of chloroform in

tooth past es an d of zinc, and t he ident ificat ion a nd det erm inat ion of phenosulfonic acid const itut esa first st ep in this direction;

Whereas the measures laid down in the present Directive are in conformity with the opinion of

th e Committee on t he a dapt at ion of Directive 76/768/EEC to technical progress,

HAS ADOPTED THIS DIRE CTIVE:

Arti cle 1

Member States shall take all necessary steps to ensure that, in the official testing of cosmetic

products: the sampling,

the laboratory prepa rat ion of test samples,

t h e id en t ifica t ion a nd det er m in a tion of fr ee sod iu m a nd pot a ss iu m hyd roxid es ,

t h e id en t ifica t ion a n d d et er m in a t ion of oxa lic a cid a nd a lk a lin e s alt s in h a ir -ca re

products,

t he det er min at ion of ch lor ofor m in toot hpa st es,

the determinat ion of zinc,

(1) OJ No L 262, 27.9.1976, p.169.

(2) OJ No L 192, 31.7.1979, p.35.


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t he iden tifica tion an d det er min at ion of ph en olsu lfon ic a cid

ar e perform ed in accorda nce with th e meth ods described in th e Annex.

Arti cle 2Member States shall bring into force the laws, regulations or administrative provisions necessary

to comply with t his Directive not lat er t ha n 31 December 1982.

They sha ll fort hwith inform th e Commission ther eof.

Arti cle 3

This Directive is addr essed to the Member Sta tes.

Done a t Brussels, 22 December 1980.

For the Commission

Richa rd BURKE

Member of th e Comm ission


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ANNEX

I. SAMP LING OF COSMETIC P RODUCTS

1. SCOP E AND FIELD OF AP P LICATION

The pr ocedur e for t he sa mpling of cosmet ic products is described with a view to th eir

an alysis in t he various labora tories.

2. DEFINITIONS

2.1 Basic sample:

a u nit t aken from a batch offered for sa le.

2.2 Total sample:

the sum of all the basic samples having the sa me batch nu mber.

2.3 Laboratory sam ple:

a r epresenta tive fra ction of the t otal sa mple tha t is to be ana lyzed in t he individual

laboratories.

2.4 Test port ion:

a r epresent at ive port ion of th e labora tory sample tha t is required for one ana lysis.

2.5 Container:

the a rt icle tha t conta ins the pr oduct an d is in cont inuous direct conta ct with it .

3. SAMP LING P ROCEDURE

3.1 Cos m et ic p rod uct s sh a ll be sa m ple d in t h eir or igin a l con t a in er s a nd for wa r de d t o t h e

an alytical laborat ory un opened.

3.2 F or cos m et ic p r od uct s wh ich a r e p la ce d on t he m a r ket in bu lk or r et a ile d in a

conta iner different from the original ma nufactur ers pa ck, appropriate inst ructions

for sa mpling at the point of use or sa le should be issued.

3.3 Th e n u m be r of ba s ic s a mp le s r equ ir ed for t h e p re pa r a t ion of t h e la bor a t or y s a m pleshall be determined by the analyt ical method and the number of analyses to be

perform ed by each labora tory.

4. SAMP LE IDENTIFICATION

4.1 S am ple s s ha ll be bot h sea led wh er e ta k en a n d id en t ifie d, in a ccor da n ce wit h t he r ule s

in force in t he relevant Member Sta te.

4.2 E a ch ba sic s am ple t ak en s ha ll be la belled wit h t he followin g in for m at ion :

n a me of t h e cos met ic p rod uct ,

d at e, t im e an d pla ce of s am plin g,

name of the person responsib le for tak ing the sample ,


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n am e of t he in spect or at e.

4.3 A r ep or t on t h e s a mp lin g s ha ll be dr a wn u p in a ccor da n ce wit h t he ru le s in for ce in

the relevant Member Sta te.

5. STORAGE OF SAMP LES

5.1 Ba sic s am ples m u st b e s tor ed in a ccor da n ce wit h t h e m a nu fa ct u rer s in st r uct ion s

appear ing on t he label if an y.

5.2 U n le ss ot h er con dit ion s a r e s pe cifie d, la bor a t or y s a mp les s ha ll be st or ed in t h e da r k

at between 10 an d 25C.

5.3 Ba sic s am ples m u st not be op en ed u n til t h e a n alys is is a bou t to begin .

II. LAB ORATORY P REP ARATION OF TES T P ORTION S

1. GENERAL

1.1 Wh er e p os sible th e a n a lys is sh a ll be ca r r ie d ou t on ea ch ba s ic s a m ple . I f t h e ba s ic

sample is too small, the minimum number of basic samples should be used. They

should first be mixed together thoroughly before t aking t he t est portion.

1.2 Op en t he con t a in er , u n de r a n in er t ga s if s o s pe cifie d in t he a n a lyt ica l m et h od a n d

withdraw the number of test portions required as quickly as possible. The analysis

should then proceed with the least possible delay. If the sample has to be preserved

the conta iner should be resealed under an inert gas.

1 .3 Cosmet ic p rod u ct s may b e p rep ared in liq uid or solid forms or in a semi-solid form. I f

separation of an initially homogeneous product occurs it should be re-homogenized

before t aking t he test port ion.

1.4 I f t h e cos m et ic p r od u ct is pu t u p for s a le in a sp ecia l w ay, a s a re su lt of w hich it

cannot be treated in accordance with these instructions, and if no provision is made

for the relevant methods of examination an original procedure may be adopted,

provided that it is set out in writing as pa rt of th e ana lysis report.

2. LIQUIDS

2.1 Th es e m a y occu r in t h e for m of p r od uct s su ch a s s olu t ion s in oil, in a lcoh ol, an d in

water, toilet waters, lotions or milks, and may be packed in flasks, bottles, ampoules

or t ubes.

2.2 Withdrawal of the tes t port ion:

shake the con tainer vigorously before opening,

open th e con ta iner ,

pour a few mill il it res of the liquid into a test-tube for visual examination of i ts

chara cter for t he pur pose of ta king the t est-port ion,

r es ea l t he con ta in er , or

wit h dr a w t h e r equ ir ed t es t por t ion s,

r es ea l t h e con t ain er ca r efu lly.


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3. SEMI-SOLIDS

3.1 Th es e m a y occu r in t h e for m of p rod uct s su ch a s pa s tes , cr ea m s, s tiff e mu ls ion s a nd

gels an d ma y be packed in tu bes, plast ic bott les or jar s.

3.2 Withdrawal of the test por t ion , either :

3 .2 .1 n ar row-n eck ed con ta in e rs . Exp el a t least t h e fir s t cent imet re of t h e prod u ct . Ext ru de

the test port ion an d reseal the conta iner immediat ely.

3 .2 .2 wide-necked containers . Scrape the surface even ly to remove the top layer . Take ou t

the test port ion an d reseal the conta iner immediat ely.

4. SOLIDS

4 .1 These may occu r in t h e form of p rod u ct s su ch as loose p owder s, comp acted p owders,

sticks and ma y be packed in a wide var iety of container s.

4.2 Withdrawal of the test por t ion , either :

4 .2.1 loose powder - sh ak e vigorou s ly be fore u n s top per in g or o pen in g. Op en an d r emove

the test port ion.

4 .2 .2 Comp act p owder or s t ick - r emove th e su r face layer by even scrap in g. Take th e t e st

portion from underneath.

5. P RODUCTS IN P RESSURIZED P ACKAGES (ae ros ol

disp en se rs)

5 .1 Th ese p rod u ct s a r e d efin ed in Ar t icle 2 of Cou n cil D irect ive 7 5/3 24 /EEC of 2 0 May

1975(1).

5.2 Test port ion:

After vigorous shaking, a representative quantity of the contents of the aerosol

dispenser are t ransferred with the aid of a sui table connector (see for example

Figure1: in specific cases the analytical method may require the use of other

connectors) into a plastic-coated glass bottle (Figure 4) fitted with an aerosol valve

but n ot fitted with a dip tu be.

During the t ran sfer t he bott le is held valve downwards. This t ra nsfer renders t he

cont ent s clear ly visible corr esponding t o one of the following four cases:

5 .2 .1 An aerosol product in the form of a homogeneous solu t ion for d irect analys is .

5 .2 .2 An aerosol p roduct consis t ing of two l iqu id phases. Each phase can be analyzed after

the lower phase has been separated into a second transfer bottle. In this case the first

transfer bottle is held valve downwards. In such a case this lower phase is often

aqueous and devoid of propellent (e.g. butane/water formulation).

5.2.3 An a e r os ol pr od u ct con t a in in g a p ow de r in s u sp en s ion . T he liq uid ph a s e ca n b e

an alyzed after r emoval of the powder.

5 .2.4 A foam or cream prod u ct . F i r st weigh exact ly i nto t h e t r an sfe r bot t l e 5 t o 1 0g of

2-methoxyethanol. This substance prevents foam from forming during the degassing

operation and it is then possible to expel the propellent gases without loss of liquid.

(1) OJ No L 147, 9. 6. 1975, p. 40.


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5.3 Accessories

The connector (Figure 1) is made of duralumin or brass. It is designed to fit to

different valve systems via a polyethylene adaptor. It is given as an example: other

connectors may be used. (See Figures 2 and 3).

The transfer bottle (Figure 4) is made of white glass coated on the outside with a

protective layer of tr an spar ent p lastic ma ter ial. It holds 50 to 100ml. It is fitted with

an aer osol valve without a dip tube.

5.4 Method

In order that enough of the sample may be transferred, the transfer bottle must be

purged of air. For this purpose, introduce through the connector about 10ml of

dichlorodifluoromet ha ne or bu ta ne (depending on th e aer osol product to be examin ed)

and then degas completely until the liquid phase disappears, holding the transfer

bott le with t he valve upper most. Remove the connector. Weigh t he t ra nsfer bottle (a

grams). Vigorously shake the aerosol dispenser from which the sample is to be taken.

Attach t he connector t o the valve on t he sa mple aer osol container (valve upwar ds), fit

the transfer flask (neck downwards) to the connector and press. Fill the transfer

bottle t o about two thirds full. If the tr ans fer ceases prem at urely owing to pressure

equalization, it can be resumed by chilling the transfer bottle. Remove the connector,

weigh th e filled bott le (b gra ms ) and det erm ine t he weight of aer osol sam ple

tra nsferred, m 1 (m 1 = b - a).

The sam ple thus obtained can be used:

1. for a nor m al ch em ica l a n alys is ;

2 . for an analysis of the volat ile const i tuents by gas chromatography.

5.4.1 Chem ica l a na lysi s

Holding the transfer bottle valve upwards, proceed as follows:

d egas . I f t h e d egass in g op era t ion gives rise to foaming, use a transfer bottle

into which an exactly-weighed quantity (5 to 10 g) of 2-methoxyethanol has

previously been intr oduced with a syringe thr ough t he connector,

complete the removal of the volat i le const i tuents without loss by shaking in a

water bath ma inta ined at 40C. Detach th e connector,

reweigh the transfer bott le (c grams) in order to determine the weight of the

residue, m 2 (m 2= c - a).

(NB: When calculating the weight of the residue, deduct the weight of any2-methoxyethanol used.)

open the t ransfer bot t le by removing the valve,

dissolve the residue completely in a known quanti ty of an appropriate solvent ,

perform the des ired determinat ion on an a liquot .

Formu las for t he calculat ion a re:

R =r x m

m

2

1

and Q =R x P

100,

where:

m1 = mass of aerosol t ak en in to t h e t r an sfe r bot t le ;

m2 = m a ss of r es id ue a ft er h ea t in g a t 40C;


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r = percen tage of the pa r t icu lar substance in m2 (deter mined according

to the appr opriat e meth od);

R = percentage of the part icular substance in the aerosol as received;

Q = total mass or the part icular substance in the aerosol dispenser;

P = net mass of init ial aerosol dispenser (basic sample).

5.4.2 Analys is o f vola t i le const i tuents by ga s chroma tograp hy

5.4.2.1 Pr inciple

Using a gas-chromatography syringe, remove an appropriate quanti ty from the

tr an sfer bottle. Then inject t he cont ents of the syringe into the gas chromat ograph .

5.4.2.2 Accessor ies

Series A2 precision s am pling gas-chr omat ogra phy syr inge 25l or 50l (Figure 5)

or equivalent. This syringe is equipped with a slide valve at the needle end. The

syringe is connected to the transfer bott le by a connector at the bott le and a

polyethylene tu be (length 8 mm , inter na l diam eter 2,5 mm) at t he syringe.

5.4.2.3 Method

After an appropriate quantity of aerosol product has been taken into the transfer

bottle, fit the conical end of the syringe to the transfer bottle as described in 5.4.2.2.

Open th e valve and a spirate a s uitable quan tity of liquid. Elimina te gas bubbles by

operating the plunger several times (chill the syringe if necessary). Close the valve

when the syringe contains the appropriate quantity of bubble-free liquid and detach

the syringe from the transfer bottle. Fit the needle, insert the syringe into the gas

chromatograph injector, open t he valve an d inject.

5.4.2.4 In ternal standard

If an intern al sta ndar d is required, it is intr oduced into the t ran sfer bottle (by means

of an ordinary glass syringe using a connector).


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III. D ETE RMIN ATION AN D ID EN TIF ICATION OF F RE E

SODIU M AND P OTASS IUM HYDR OXIDES


The method specifies the procedure for identifying cosmetic products containing

significant amounts of free sodium and/or potassium hydroxides and for the

determina tion of such free sodium and/or potassium hydroxides in h air str aightener

prepar at ions an d nail cuticle solvent prepa ra tions.

2. DEFINITION

The free sodium and potassium hydroxide is defined by the volume of standard acid

required to neut ralize the pr oduct un der specified conditions, th e resulting qua ntity

being expressed as % m/m free sodium hydroxide.

3. P RINCIP LE

The sample is dissolved or dispersed in water and titrated with standard acid. The

pH value is recorded concurrently with addition of acid: for a simple solution of

sodium or potassium hydroxides the end point is a clear cut maximum rate of change

of recorded pH va lue.

The simple titr at ion curve m ay be obscured by the pr esence of:

(a) ammonia and other weak organ ic bases , which have themselves a ra ther f la t

titra tion curve. Amm onia is removed in t he m ethod by evaporation at reduced

pressure but at r oom tempera ture;

(b) sal ts of weak acids, which may give r ise to a t i t rat ion curve with several points

of inflection. In such cases only the first part of the curve to the first of these

points of inflection corresponds to the neutralization of hydroxyl ion coming

from free sodium or potassium hydroxide.

An alter na tive procedur e for t itra tion in a lcohol is given wher e excessive interferen ce

from salts of weak inorganic acids is indicated.

Whilst the theoretical possibility exists that other soluble strong bases, e.g. lithium

hydroxide, quaternary ammonium hydroxide, could be present giving rise to the high

pH, th e pres ence of these in t his t ype of a cosmetic product is highly un likely.

4. IDENTIFICATION

4.1 Reagent s

4.1.1 S t a n da r d a lk a lin e bu ffe r solu t ion p H 9,1 8 a t 2 5C : 0 ,0 5 M sod iu m t e t r a bor a t e

decahydrate.

4.2 Apparatus

4.2.1 Usua l la bor atory gla ssware

4.2.2 pH meter

4.2.3 Glass membrane elect rode

4.2.4 St an da r d ca lom el r efer en ce elect rode.


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4.3 Procedure

Calibrate the pH meter with the electrodes using the standard buffer solut ion.

Prepare a 10% solution or dispersion of the product to be analyzed, in water, and

filter. Measur e the pH . If the pH is 12 or over a quant itative determinat ion m ust be

carried out.

5. DETERMINATION

5.1 Titrat ion in aqueous med ium

5.1.1 Reagent

5.1.1.1 S ta n da r d 0,1 N hydr och lor ic a cid

5.1.2 Ap p a ra tus

5.1.2.1 U su al la bor at or y gla sswa re

5.1.2.2 p H m et er pr efer a bly wit h r ecor der

5.1.2.3 Gla ss mem br an e elect rode

5 .1.2.4 S tan d ard ca lomel r e fe ren ce e lect rod e.

5.1.3 Procedure

Weigh accurately into a 150-ml beaker a test portion of between 0,5 and 1,0g. If

am monia is present a dd a few ant i-bumping granules, place the beak er in a vacuum

desiccator, evacuate using a water pump until the odour of ammonia is no longer

detectable (about th ree hours).

Add 100 ml water , dissolve or disperse the residue and t i t rate with the 0,1N

hydr ochloric acid solution (5.1.1.1) recordin g th e cha nge in pH (5.1.2.2).

5.1.4 Calcula t ion

Identify the points of inflection on the titration curves. Where the first point of

inflection occurs a t a pH below 7 th e sam ple is free of sodium or potas sium hydr oxide.

Where t here ar e two or m ore points of inflection in th e curve only the first is relevant.

Note th e volum e of titra nt to this first point of inflection.

Let V represent t his volume of titr an t, in ml,

M represent th e weight of th e test portion, in gra ms.

The content of sodium and/or potassium hydroxides in the sample expressed as

%m/m of sodium h ydroxide is calcula ted u sing th e form ula :

% = 0, 4V

M

The situation may arise in which, despite indications of the presence of a significant

quantity of sodium and/or potassium hydroxides, the titration curve fails to show a

distinct point of inflection. In such a case the determination should be repeated in

isopropanol.

5.2 Titrat ion in i sopropan ol

5.2.1 Reagents

5.2.1.1 Isopropanol

5 .2.1.2 S tan d ard 1 ,0 N aqu eou s h yd roch lor ic acid


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5 .2 .1 .3 0 ,1 N hydroch lor ic acid in i sopropanol p repared immediate ly before use by di lu t ing

th e 1,0 N a queous h ydrochloric acid with isopropan ol.

5.2.2 Ap p a ra tus

5.2.2.1 U su al la bor a tor y gla ss wa re

5.2.2.2 pH me ter pr efer a bly wit h r ecor der

5.2.2.3 Gla ss mem br an e elect rode

5 .2 .2 .4 S tan dard ca lomel r efe rence e lect rod e.

5.2.3 Procedure

Weigh accurately into a 150-ml beaker a test portion of between 0,5 and 1,0g. If

ammonia is present add a few antibumping granules, place the beaker in a vacuum

desiccator, evacuate using a water pump until the odour of ammonia is no longer

detectable (about th ree hours).

Add 100 ml isopropanol, dissolve or disperse the residue and titrate with the 0,1N

hydrochloric acid in isopropanol (5.2.1.3) recording the change in apparent pH

(5.2.2.2).

5.2.4 Calcula t ion

As in 5.1.4. The first point of inflection is at an appa ren t pH of about 9.

5.3 Repeatabi l i ty(1)

For a sodium or potassium hydroxide content in the range of 5%m/m as sodium

hydroxide, the difference between the results of two determinations carried out in

par allel on t he sa me sam ple should not exceed an absolute value of 0,25%.

IV. D ETE RMIN ATION AN D ID EN TIF ICATION OF

OXALIC ACID AND ITS ALKALINE SALTS IN

HAIR-CARE P RODU CTS


The method described below is suitable for the determination and identification of

oxalic acid and its alkaline salts in hair-care products. It can be used for colourless

aqueous/alcoholic solutions and lotions which contain about 5% of oxalic acid or an

equivalent quan tity of alka line oxalate.

2. DEFINITION

The content in oxalic acid and/or its alkaline salts determined by this method is

expressed as a percenta ge by mass (m/m) of free oxalic acid in th e sa mple.

3. P RINCIP LE

After removal of any anionic surface-act ive agents present with p-toluidine

hydrochloride, th e oxalic acid an d/or t he oxalates ar e precipitated as calcium oxalate,

(1) See ISO/DIS 5725.


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whereupon the solution is filtered. The precipitate is dissolved in sulphuric acid and

titrat ed against potassium perman ganate.

4. REAGENTS

All rea gents s hould be of ana lytical pu rity

4.1 5% (m/m) a mmonium aceta te solut ion

4.2 10% (m /m ) ca lciu m chlor ide solu tion

4.3 95% (V/V) ethanol

4.4 carbon tet rach lor ide

4.5 diethyl ether

4.6 6,8% (m /m ) p-t olu idin e dih ydr och lor ide solu tion

4.7 0,1N potassium permanganate solu t ion

4.8 20% (m/m) su lphur ic acid

4.9 10% (m/m) hydroch lor ic acid

4.10 Sodium aceta te t r ihydrate

4.11 Acet ic acid glacia l

4.12 Sulphur ic acid (1:1)

4.13 Sa tu ra ted ba riu m hydr oxide solut ion.

5. AP P ARATUS

5.1 Separa t ing funnels, 500ml

5.2 Beakers, 50 ml and 600ml

5.3 Glass filter crucibles, G-4

5.4 Measur ing cylinders, 25ml a nd 100m l

5.5 Pipet tes, 10ml

5.6 Suct ion flasks, 500ml

5.7 Water-jet pump

5.8 Therm ometer gra duated from 0 to 100C

5.9 Ma gnet ic st ir rer with heat ing element

5.10 Ma gn et ic st ir rin g r ods, t eflon -coa ted

5.11 Buret te, 25ml

5.12 Conical flasks, 250ml.

6. P ROCEDURE

6.1 We igh ou t 6 t o 7 g of t h e s a mp le in t o a 50-m l be ak er , b rin g t h e p H t o 3 w it h dilu t e

hydrochloric acid (4.9) and wash into a separating funnel with 100 ml of distilled

water. Add successively 25ml of ethanol (4.3), 25ml of p-toluidine dihydrochloride


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16

solution (4.6) and 25 to 30ml of carbon tetrachloride (4.4) and shake the mixture

vigorously.

6.2 Aft er s ep ar a t ion of t h e p ha se s, r em ove th e lower (or ga n ic) p ha se re pea t t h e

extraction, using the r eagents m entioned in 6.1, and again r emove the organic phase.

6.3 Wa sh t h e a qu eou s solu t ion in t o a 600-m l be ak er a n d r em ove a ny ca r bon t et r a ch lor id e

still present by boiling the solution.

6 .4 Ad d 50 ml of ammon iu m ace t at e solu t ion (4.1 ), b rin g th e solu t ion to t h e b oil (5 .9 ) an d

stir 10ml of hot calcium chloride solution (4.2) into the boiling solution; allow the

precipitat e to settle.

6.5 Ch eck t h a t pr ecip it a t ion is com plet e by a dd in g a fe w d rop s of ca lciu m ch lor id e

solution (4.2), allow to cool to room temperature and then stir in 200ml of ethanol

(4.3); (5.10) leave t o stan d for 30 m inu tes.

6.6 F ilt er t h e liqu id t hr ou gh a gla s s filt er cr u cible (5.3), t r a n sfe r th e pr ecip it a t e w it h a

small quantity of hot water (50 to 60C) into the filter crucible and wash theprecipitat e with cold water .

6.7 Wa s h t h e p re cip it a t e five tim es wit h a lit t le et h a nol (4.3) a n d t h en five tim e wit h a

little diethyl ether (4.5) and dissolve the precipitate in 50ml of hot sulphuric acid

(4.8) by drawing th e latt er t hrough th e filter crucible under reduced pressur e.

6.8 Tr a n sfe r t he solu t ion w it h ou t los s in t o a con ica l fla s k (5.11) a n d tit r a te a ga in st

pota ssium perma ngana te s olution (4.7) unt il a light pink colour at ion occurs.

7. CALCULATION

The content of the sample expressed as oxalic acid percentage by mass is calculatedfrom t he form ula

% oxalic a cid =A x 4,50179 x 100

E x 1000

in which:

A is th e con su mpt ion of 0,1N pot assiu m perm an gan a te mea sur ed in

accorda nce with 6.8;

E is the test quant ity of sample in grams (6.1);

4,50179 is t he con ver sion fa ct or for oxa lic a cid.

8. REP EATABILITY(1)

For an oxalic acid content of about 5% the difference between the results of two

determinations in paral lel carried out on the same sample should not exceed an

absolut e va lue of 0,15%.

9. IDENTIFICATION

9.1 Principle

Oxalic acid and/or oxalates are precipitated as calcium oxalate and dissolved in

sulphuric acid. To the solution is added a little potassium permanganate solution,



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which turns colourless and causes the formation of carbon dioxide. When the

resultant carbon dioxide is passed through a barium hydroxide solution, a white

precipitat e (milkiness) of barium carbonate is form ed.

9.2 Procedure

9 .2 .1 Trea t a p or t ion of t h e samp le t o b e an a lyzed as d escr ib ed in sect ion 6 .1 t o 6 .3 ; t h is

will remove any deter gents presen t.

9 .2 .2 Add a spatu la t ipfu l of sod ium aceta te (4 .10) to about 10ml of the solu t ion ob tained

in accordance with 9.2.1 and acidify the solution with a few drops of glacial acetic

acid (4.11).

9 .2.3 Ad d 10 % ca l cium ch lor ide solu t ion (4.2 ) an d fi lt e r . Di ssolv e t h e ca l cium o xa la t e

precipita te in 2m l of sulphu ric a cid (1:1) (4.12).

9.2.4 T r a ns fe r t h e s olu t ion i nt o a t e st t u b e a n d a dd dr op -w is e a b ou t 0 ,5 m l of 0 ,1 N

potassium perm an gana te s olution (4.7). If oxalat e is presen t, th e solut ion loses colour

at first gradua lly and t hen r apidly.

9.2.5 I m me dia t ely a ft er a d din g t h e pot a s siu m p er m a n ga n a t e, pla ce a n a pp r op r ia t e gla s s

tube with stopper over the test tube, heat the contents slightly and collect the carbon

dioxide formed in a saturated barium hydroxide solution (4.13). The appearance,

after three to five minutes, of a milky cloud of barium carbonate indicates the

presence of oxalic acid.

V. DETERMINATION OF CHLOROFORM IN

TOOTHPASTE


This method is used for the determination of chloroform in toothpaste by gas

chromat ograph y. This meth od is suit able for t he det erm inat ion of chloroform at levels

of 5% or less .

2. DEFINITION

The chloroform content determined by this method is expressed as a percentage by

ma ss of th e product.

3. P RINCIP LE

The toothpaste is suspended in a dimethylformamide/methanol mixture to which is

added a known quantity of acetonitrile as internal standard. After centrifuging, a

portion of the liquid phase is subjected to gas chromatography and the chloroform

cont ent calculat ed.

4. REAGENTS

All rea gents s hould be of ana lytical pu rity.

4.1 P or apa k Q, Ch rom osor b 101 or equ iva len t, 80 t o 100 m es h

4.2 Aceton it r ile


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4.3 Chloroform

4.4 Dimethylformamide

4.6 Interna l standard solu t ion

Pipette 5ml of dimethylform am ide (4.4) into a 50-ml st an dar d flask a nd add about

300mg (M mg) of acetonitrile, accurately weighed. Make up to the mark with

dimethylform am ide and mix.

4.7 S olu t ion for t h e d et e rm in a t ion of r e la t ive r es pon s e fa ct or . P ip et t e e xa ct ly 5 m l of

internal standard solution (4.6) into a 10-ml standard flask and add about 300mg

(M1 mg) of ch loroform, accurate ly weighed . Make up to the mark wi th

dimethylform am ide and mix.

5. AP P ARATUS AND EQUIP MENT

5.1 Analyt ica l ba lance.

5.2 Ga s ch rom at ogr aph , wit h fla me ion iza tion det ect or .

5 .3 Micro-syr in ge with a cap acity of 5 t o 1 0 l an d gradu a t ion of 0,1 l.

5.4 Bu lb pipet tes wit h ca pa cit ies of 1, 4 a nd 5 m l.

5.5 Volumetr ic flasks, 10 and 50 ml.

5.6 Te st t u be s, a pp roxim a t ely 20 m l, w it h scr ew ca p s, S ovir el Fr a n ce No20 or

equivalent . The crew cap ha s an intern al sealing plate coat ed on one side with teflon.

5.7 Cent r ifuge.

6. P ROCEDURE

6.1 Suitable gas chromatography condi t ions

6.1.1 Column mater ia l: glass

length : 150 cm

internal diameter: 4 mm

externa l diameter : 6 mm.

6 .1.2 Pack th e colu mn with Porap ak Q, Ch romosorb 10 1 or equ iva lent 8 0 to 10 0 mesh (4 .1)

with th e aid of a vibrat or.

6 .1 .3 Detector , flame ion izat ion : ad jus t it s sens it ivi ty so that when 3 l of solu t ion 4 .7 i s

injected, the height of the acetonitr i le peak is about three quarters ful l-scale

deflection.

6.1.4 Gases:

Carr ier, nitr ogen, flow r at e 65ml/min.

Auxiliary: adjust th e flow of gases to th e det ector so tha t th e flow of air or oxygen is

five to 10 times t hat of the h ydrogen.

6.1.5 Tem p era tures :

in jector block 210C

det ect or block 210C


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19

column oven 175C.

6.1.6 Cha r t sp eed :

6.2 Sample preparat ion

Take the sample for analysis from an unopened tube. Remove one third of the

content s, replace the cap on t he tu be, mix carefully in t he tu be and th en ta ke the test

portion.

6.3 Determinat ion

6 .3 .1 Weigh ou t , in to a screw-cap ped tu be (5.6) t o t h e n earest 1 0mg, 6 t o 7 g (Mog) of the

tooth past e prepa red in a ccorda nce with section 6.2, an d add t hree sm all glass bea ds.

6.3.2 P ip et t e exa ct ly 5m l of th e in t er n a l st a n da r d s olu t ion ( 4.6 ), 4m l of

dimethylformamide (4.4) and 1ml of methanol (4.5) into the tube, close the tube and

mix.

6 .3 .3 Sh ake for h a lf an h ou r with a mech an ica l sh aker an d cen tr ifu ge th e closed tu be for15 minutes, at such a speed as to produce a clear sepa ra tion of th e phases.

Note: It occasionally happens that the liquid phase is still cloudy after centrifuging.

Some improvement can be obtained by adding 1 t o 2g of sodium chloride t o the

liquid pha se, allowing to sett le an d r ecentr ifuging.

6.3.4 Inject 3 l of this solut ion (6.3.3) under the condit ions described in sect ion 6.1. Repeat

this operation. For the conditions described above, the following retention times can

be given a s guide values:

methanol approximately one minute

aceton it r ile approximately 2,5 minutes

ch loroform approximately six minutes

dimethylformamide >15 minutes

6.3.5 Determina t ion of the rela t ive response factor

Inject 3l of solution 4.7 for the determination of this factor. Repeat this operation.

Determ ine the r elative response factor da ily.

7. CALCULATIONS

7.1 Calculat ion of the re lat ive respon se

7 .1.1 Measu re t h e h e igh t an d th e wid th a t h a lf h e igh t of t h e ace ton it r ile and ch loroform

peaks a nd calculate t he a rea of both peaks, using the formu la: height x width a t h alf

height.

7 .1.2 Dete rmin e th e a rea of t h e ace ton it r ile an d ch loroform peak s in t h e ch romatograms

obtained in accordance with section 6.3.5 and calculate the relative response fs with

th e a id of th e following form ula :

fs= As Mi

Ms Ai

As 1 10 M

Ai M1

=

in which:

fs = th e rela tive resp onse factor for chloroform;

As = the area of the chloroform peak (6.3.5);


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Ai = the area of the acetonitr ile peak (6.3.5);

Ms = the quanti ty of chloroform in mg per 10ml of the solut ion referred

to in section 6.3.5 (= M1);

Mi = th e quan tity of acetonitrile in mg per 10ml of th e solut ion referredto in s ection 6.3.5 (= 1/10 M).

Calculat e the mea n of the r eadings obtained.

7.2 Calculat ion of the ch loroform con tent

7.2 .1 Calcu la t e in accord an ce with i t em 7 .1 .1 t h e a rea of t h e ch loroform an d ace ton it r ile

peaks of th e chromat ogram s obtained by th e procedure described in section 6.3.4.

7.2 .2 Calcu l a t e t h e ch loroform con ten t in t h e t ooth p as t e with t h e a id of t h e fol lowin g

formula:

%X =As Mi

f M Ai

100 %As M

f Ai M 100s sx s 0

=

in which:

% X = th e chloroform cont ent of th e toothpa st e express ed by ma ss;

As = th e ar ea of th e chloroform pea k (6.3.4);

Ai = th e ar ea of th e acetonit rile peak (6.3.4);

Msx = th e ma ss in mg of th e sam ple referr ed to in sect ion 6.3.1

(=1000Mo)

Mi = th e quan tity of acetonitrile in mg per 10 ml of th e solution obtain ed

in a ccordan ce with section 6.3.2 (1/10 M).

Calculate th e mea n of th e levels found an d express th e resu lt to an accur acy of within

0,1%.


For a chloroform content of about 3%, the difference between the results of two

determinations in paral lel carried out on the same sample should not exceed an

absolut e va lue of 0,3%.

VI. DETERMINATION OF ZINC


This method is suitable for the determination of zinc present as chloride, sulphate or

4-hydroxybenzenesulphonate, or as an association of several of these zinc salts, in

cosmetics.

2. DEFINITION

The zinc content of the sample is determined gravimetrically as the bis(2-methyl-8-

quinolyl oxide) and is expressed as percenta ge by mass of zinc in t he sa mple.

(1 ) See ISO/DIS 5725.


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3. P RINCIP LE

Zinc present in solution is precipitated in an acid medium as zinc bis(2-methyl-8-

quinolyl oxide). After filtration the precipitate is dried and weighed.

4. REAGENTS


4.1 25% (m/m) concent ra ted ammonia ; d20

4=0,91

4.2 Glacia l acet ic acid

4.3 Ammonium aceta te

4.4 2-Methylquinolin-8-ol

4.5 6% (m/v) ammonia solu t ion

Transfer 240g of concentrated ammonia (4.1) into a 1000-ml standard flask, make

up to the m ark with distilled water a nd mix.

4.6 0,2M ammonium aceta te solut ion

Dissolve 15,4 g of ammonium acetate (4.3) in distilled water, make up to the mark in

a 1000-ml sta nda rd flask an d mix.

4.7 2-Methylquinolin-8-ol solut ion

Dissolve 5 g of 2-methylquinolin-8-ol in 12 ml of glacial acetic acid and transfer with

distilled water into a 100-ml standard flask. Make up to the mark with distilled

water a nd mix.


5.1 Standard flasks, 100 and 1000ml

5.2 Beakers, 400 ml

5.3 Measur ing cylinders, 50 and 150ml

5.4 Graduated pipet tes, 10 ml

5.5 Glass filter crucibles G-4

5.6 Vacuum flasks, 500 ml

5.7 Water-jet pump

5.8 Therm ometer gra duated from 0 to 100C

5.9 D es icca t or w it h a s u it a ble de sicca n t a n d hu m id it y in dica t or . e .g. silica -ge l or

equivalent

5.10 Dr yin g oven regu la ted t o a tem per a tu re 150 2C

5.11 pH meter

5.12 Hot pla te

5.13 F ilt er pa per , Wh at ma n No 4 or equiva lent .


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6. P ROCEDURE

6 .1 Weigh ou t in to a 40 0-ml b eak er , 5 t o 1 0 g (M grams), con ta in in g abou t 50 to 1 00 mg

of zinc, of th e sam ple to be ana lyzed add 50ml of distilled wat er a nd m ix.

6 .1 .1 F ilt er , w ith t h e a id of a vacu u m pu mp if n ecessa ry, an d r et a in t h e filt r a t e.

6 .1 .2 Rep ea t t h e ext r act ion s t ep with a fur th er 5 0ml of d is t illed wa te r. F ilt e r an d combin e

the filtra tes.

6.2 F or eve ry 1 0 m g of zin c p re se nt in t he s olu t ion (6.1.2) a d d 2 m l of t h e

2-met hylquinolin-8-ol solut ion (4.7) an d mix.

6.3 Dilu t e t h e m ixt u r e wit h 150 m l of d is tilled wa t er , br in g t h e t em pe ra t u re of t h e

mixture up to 60C (5.12) and add 45 ml of 0,2M ammonium acetate solution (4.6),

stirr ing const an tly.

6 .4 Ad ju s t th e pH of t h e solu t ion to 5 ,7 t o 5 ,9 , w ith 6% ammon ia solu t ion (4 .5 ), s t ir r in g

const an tly; use a pH met er to measu re th e pH of th e solution.

6.5 Allow t h e s olu t ion t o s t a nd for 30 m in u t es . F ilt er wit h t he aid of a wa t er -je t p um p

through a G-4 filter crucible which has been dried beforehand (150C) and weighed

after cooling (M0 grams), and wash the precipitate with 150ml of distilled water at

95C.

6 .6 P lace t h e cru cible in a dryin g oven r egu la t ed to 1 50 C an d d ry for on e h ou r .

6 .7 Remove the cru cib le from th e d ryin g oven , p lace it in a desicca tor (5 .9 ) an d , wh en it

ha s cooled to room tem pera tur e, determ ine the ma ss (M1grams).

7. CALCULATION

Calculate the zinc conten t of the sam ple as a percentage by mass (% m/m) with th e

aid of th e following form ula :

% zinc = (M M ) x 17, 12

M

1 0

in wh ich

M = t he m as s in gr am s of t he sa m ple t a ken in accor da nce wit h 6.1;

M0 = the mass in grams of the empty and dry fi lter crucible (6 .5);

M1 = the mass in grams of the fi lter crucible with precipi tate (6 .7).


For a zinc content of about 1% (m/m), the difference between the results of two

determinations in parallel on the same sample should not exceed an absolute value of

0,1%.

(1) I SO /DI S 5 72 5.


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VII. D ETE RMIN ATION AN D ID EN TIF ICATION OF

4-HYDROXYBENZENESULPHONIC ACID


T h i s m e t h o d i s s u i t a b l e f o r t h e i d e n t i f i c a t i o n a n d d e t e r m i n a t i o n o f

4-hydroxybenzenesulphonic acid in cosmetic products such as aerosols and face

lotions.

2. DEFINITION

The 4-hydroxybenzenesulphonic acid content determined in accordance with this

method is expressed as a percentage by mass of anhydrous zinc 4-hydroxy-

benzenesulphonate in th e product.

3. P RINCIP LE

The test portion is concentrated under reduced pressure, dissolved in water and

pur ified by chloroform extr action. The deter mina tion of 4-hydroxybenzene-sulphonic

acid is car ried out iodomet rically on a n a liquot of th e filter ed aqu eous solut ion.

4. REAGENTS


4.1 36% (m /m ) con cen tr at ed h ydr och lor ic a cid (d20

4=1,18)

4.2 Chloroform

4.3 Butanol-1-ol

4.4 Glacia l acet ic acid

4.5 Potassium iodide

4.6 Potassium bromide

4.7 Sodium carbona te

4.8 Sulphanilic acid

4.9 Sodium nit r ite

4.10 0,1N potassium bromate

4.11 0,1N sodium t hiosulph at e solu tion

4.12 1% (m /v) a qu eou s solu tion of st ar ch

4.13 2% (m /v) a qu eou s s olu t ion of s odiu m ca r bon a te

4.14 4,5% (m /v) a qu eou s s olu t ion of s od iu m n it r it e

4 .1 5 0,05 % (m/v) solu t ion of d it h izon e in ch loroform

4.16 Develop ing solven t : bu tan-1-ol /g lacia l acet ic acid/water (4:1 :5 par t s by volume); after

mixing in th e separa ting funn el, discar d the lower pha se.


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4.17 Pauly r eagent

Dissolve 4,5 g of sulph an ilic acid (4.8) in 45 m l of concentr at ed h ydrochloric a cid (4.1),

while heat ing, and dilute t he solution with wat er t o 500ml. Cool 10ml of the solution

in a dish with ice-water and add, while stirring, 10 ml of cold sodium nitrite solution

(4.14). Allow the solution to stand for 15 minutes at 0C (at this temperature the

solution remains stable for one to three days) and immediately before spraying (7.5)

add 20 ml of sodium carbona te s olut ion (4.13).

4 .1 8 Ready-p rep ared ce llu lose p la t es for t h in -l ayer ch romatograp h y; format 2 0 x 2 0cm,

thickness of th e adsorbent layer 0,25 mm.


5.1 Rou nd-bot ton ed fla sk s wit h gr ou nd gla ss st opper , 100 m l

5.2 Separat ing funnel, 100ml

5.3 Con ica l fla sk wit h gr ou nd gla ss st opper , 250m l

5.4 Buret te, 25 ml

5.5 Bulb pipet tes, 1, 2 and 10ml

5.6 Gradua ted pipet te, 5 ml

5.7 Micr o-syr in ge, 10 l wit h 0,1 l gr adu at ion s

5.8 Thermometer graduated from 0 to 100C

5.9 Water bath equipped with a heat ing element

5.10 Dr yin g oven , well ven tila t ed a nd r egu la ted at 80C

5.11 Th e u su a l a pp ar a t us for ca r r yin g ou t th in -la ye r ch r om a t ogr a ph y.

6. SAMP LE P REP ARATION

In the method descr ibed below for the iden t f icat ion and determinat ion of

hydroxybenzenesulphonic acid in aerosols use is made of the residue obtained by

releasing from the aerosol can the solvents and propellants that vaporize at normal

pressure.

7. IDENTIFICATION

7 .1 With t h e a id of a micro-syr in ge (5.7) ap ply 5 l of t h e resid u e (6) or sample a t each of

six points on the starting line at a distance of 1 cm from the lower edge of the

thin-layer plate (4.18).

7.2 P la ce t he pla t e in a de ve lop in g t a n k wh ich a lr ea d y con t a in s th e d eve lop in g s olve nt

(4.16) and develop un til the solvent front ha s rea ched 15 cm from th e sta rt ing line.

7.3 Re move th e p la t e fr om t h e ba t h an d d ry a t 80C un t il n o a cet ic a cid va pou r is

perceptible. Spray t he plat e with sodium carbonat e solution (4.13) and dr y in air.

7.4 Cover on e h a lf of t h e p la t e wit h a gla ss pla t e a n d s pr a y t h e u n cover ed pa r t wit h

0,05% dithizone solution (4.15). The appearance of purplish-red spots in the

chromatogram indicat es t he pr esence of zinc ions.

7.5 Cove r t h e s pr a yed ha lf of t h e p la t e wit h a gla s s p la t e a n d s pr a y t h e ot h er h a lf w it h

Pauly reagent (4.17). The presence of 4-hydroxybenzenesulphonic acid is indicated by


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the appearance of a yellowish-brown spot with an Rf value of about 0,26 whilst a

yellow spot with an Rf value of about 0,45 in the chromatogram indicates the

pres ence of 3-hydr oxybenzenesu lphonic acid.

8. DETERMINATION

8 .1 Weigh ou t 10 g of t h e sample or r es id u e (6 ) in to a 10 0-ml rou n d-b ot tomed fla sk an d

evaporate almost to dryness under vacuum in a rotary evaporator over a water bath

kept a t 40C.

8.2 Pipet te 10,0ml (V1 ml) water into the flask and dissolve the evaporation residue (8.1)

by heating.

8.3 Qu a nt it a tively t r a ns fer t h e s olu t ion in t o a s ep ar a t in g fu n nel (5.2) a n d ext r act t h e

aqueous solution twice with 20-ml portions of chloroform (4.2). After each extraction

discar d t he chloroform ph ase.

8.4 F ilt er t h e a qu eou s s olu t ion t hr ou gh a flu t ed filt er . De pe nd in g on t he exp ect edhydroxybenzenesulph onic acid cont ent , pipette 1,0 or 2,0 ml (V2) of the filtrate into a

250-ml conical flask (5.3) an d dilut e to 75 ml with wat er.

8 .5 Add 2 ,5 ml of 36% hydroch lor ic acid (4 .1 ) and 2,5 g of potass ium bromide (4 .6 ), mix

an d bring the tem pera tur e of th e solution up to 50C with th e aid of a wa ter ba th.

8.6 F r om a b ur et t e, ad d 0,1N p ot a ss iu m br om a t e (4.10) u n t il th e solu t ion , w hich is s till

at 50C turns yellow.

8 .7 Ad d a fu r th er 3,0 ml of p ot as siu m bromate solu t ion (4.1 0), s t op per th e fla sk an d a llow

to stand for 10 minut es in a water bath at 50C.

If after 10 minutes the solution loses its colour, add another 2,0ml of potassiumbromate solution (4.10), stopper the flask and heat for 10 minutes over a water bath

kept a t 50C. Record t he t ota l quan tity of pota ssium bromate solution added (a).

8 .8 Cool t h e solu t ion to room t emp era tu re, ad d 2g of p ot as siu m iod id e (4.5) an d mix.

8 .9 T it r a t e t h e iod in e formed aga in s t 0,1 N sod iu m th iosu lp h a t e solu t ion (4 .1 1). Toward s

the end of the titration add a few drops of starch solution (4.12) as indicator. Record

the quan tity of sodium thiosulphat e used (b).

9. CALCULATION

Calculate the zinc hydroxybenzonesulphonate content of the sample or residue (6) as

a per centa ge by ma ss (% m/m) with t he a id of th e following formu la:

% m/m zinc hydroxybenzenesulphonate =(a b ) x V x 0 ,00514 x 100

m x V

1

2

in which:

a = th e t ot a l q u an t i t y i n mi ll il it r e s of 0 ,1 N p otass iu m b romate

solution a dded (8.7),

b = t he qu a nt it y in m illilit res of 0,1N sodiu m th iosu lph at e s olu tion

used for the back-titration (8.9),

m = t h e q u a n t i t y of p r od u ct or r e s id u e a n a l yz ed , e xp r e ss e d i n

milligram s (8.1),


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26

V1 = th e volum e of th e solut ion obta ined in accorda nce with 8.2,

expressed in millilitres,

V2 = th e v olu me of t h e di ssolved evap ora t ion r es idu e u sed for t h e

analysis (8.4), expressed in millilitres.

Note: In the case of aerosols, the measurement result in % (m/m) of the

residue(6) must be expressed in terms of original product. For the

purpose of this conversion, reference is made to the rules for the

sa mpling of aer osols.


For a content of about 5% zinc hydroxybenzenesulphonate, the difference between

the results of two determinations carried out in parallel on the same sample should

not exceed an absolut e value of 0,5%.

11. INTERP RETATION OF THE RESULTS

Under Council Directive 76/768/EEC relating to cosmetic products, the maximum

authorized concentration of zinc 4-hydroxybenzenesulphonate in face lotions and

d e o d o r a n t s i s 6 % ( m / m ) . T h i s f o r m u l a t i o n m e a n s t h a t b e s i d e s t h e

hydroxybenzenesulphonic acid content , the zinc content must be determined.

Multiplication of the calculated zinc hydroxybenzenesulphonate content (9) by a

factor of 0,1588 yields the minimum zinc content in % (m/m) that must theoretically

be present in the product in view of the measured hydroxybenzenesulphonic acid

content. The zinc content as actually measured gravimetrically (see the relevant

provisions) may, however, be higher, because zinc chloride and zinc sulphate may

also be used in cosm etic products.



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27

SECOND COMMISSION DIRECTIVE 82/434/EEC

Second Commission Directive 82/434/EEC 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

(As am ended by Com m ission Directive 90/ 207/ EE C of 4 April 1990)

THE COMMISSION OF THE EU ROPEAN COMMUNITIES,

Ha ving regard t o the Tr eat y establishing the Eur opean E conomic Comm unity,

Ha ving regar d to Council Directive 76/768/EE C of 27 J uly 1976 on t he a pproxima tion of th e laws

of the Member States relating to cosmetic products(1), as amended by Directive 79/661/EEC(2),

an d in par ticular Art icle 8(1) thereof,

Wherea s Directive 76/768/EE C pr ovides for th e official t esting of cosmetic products with th e a im

of ensu ring t ha t t he conditions laid down by Comm un ity provisions concerning t he composition of

cosmet ic products a re sat isfied;

Whereas all the necessary methods of analysis should be drawn up as quickly as possible;

whereas, the first st ep towards the a tta inment of this objective having already been tak en by the

definition of certain methods in Commission Directive 80/1335/EEC(3), the second step is to

consist in t he definition of meth ods for identificat ion of some oxidizing agents an d deter mina tion

of hydrogen peroxide in cosmetic hair-care products, identification and semi-quantitativedetermination of certain oxidation colorants in hair dyes, identification and determination of

nitrite, identification and determination of free formaldehyde, determination of resorcinol in

sha mpoos a nd h air lotions, an d determ inat ion of metha nol in r elation to etha nol or pr opan-2-ol;

Whereas the measures laid down in this Directive are in accordance with the opinion of the

Comm ittee on the ada pta tion of Directive 76/768/EE C to t echnical pr ogress,

HAS ADOPTED TH IS DIRECTIVE:

Arti cle 1

Member States shall take all necessary steps to ensure that, during official testing of cosmetic

products:

id en t ifica t ion of oxid izin g agent s and de t ermin a t ion of h yd rogen p eroxid e in h a ir -ca re

products,

id en t ifica t ion a n d s em i-qu a n t it a t ive d et er m in a t ion of ce rt a in oxid a tion color a n t s in

hair dyes,

ident ificat ion and determinat ion of nit r ite,

iden tifica tion an d det er min at ion of fr ee for ma ldeh yde,

(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.


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28

det er min at ion of r esor cin ol in sh am poos an d h air lot ion ,

det er min at ion of m et ha nol in r ela t ion t o et ha nol or pr opa n-2-ol

ar e perform ed in accorda nce with th e meth ods described in th e Annex.

Arti cle 2

Member States shall bring into force the laws, regulations or administrative provisions necessary

to comply with t his Directive not lat er t ha n 31 December 1983.

They sha ll fort hwith inform th e Commission ther eof.

Arti cle 3

This Directive is addr essed to the Member Sta tes.

Done a t Brussels, 14 May 1982.

For the Commission

Kar l-Heinz NARJ ES

Member of th e Com m ission


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ANNEX

I. ID EN TIFICATION OF OXID IZIN G AGEN TS AN D

DE TERMINATION OF H YDROGEN P EROXIDE IN

HAIR-CARE P RODU CTS

PURP OSE 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 th e iodomet ric determ inat ion of hydrogen per oxide it is n ecessary t o detect an d

identify any other oxidizing agents present. This identification breaks down into two

stages; the first covers the persulphates, the bromates and hydrogen peroxide and

the second covers barium peroxide.

A. ID EN TIF ICATION OF PERS ULP HATES , B ROMATES AND

HYDROGEN P EROXIDE

1. P RINCIP LE

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 m ade of two developing solvents.

2. REAGENTS


2 .1 0,5 % (m/v) aqu eou s r efe rence solu t ion s of t h e followin g comp ou n ds :

2.1.1 Sodium persu lpha te

2.1.2 Potassium persulphate2.1.3 Ammonium persu lphate

2.1.4 Potassium bromate

2.1.5 Sodium bromate

2.1.6 Hydrogen peroxide

2.2 Developin g solven t A, 80% (v/v) et ha nol

2.3 D eve lop in g s olve nt B, b en ze ne m e t ha n ol 3-m e t hyl b ut a n -1-ol w a t er

(34:38:18:10 by vol)

2 .4 Detect in g agent A, 1 0% (m/v) aqu eou s solu t ion of p ot ass iu m iod id e

2.5 Det ect in g a gen t B, 1% (m /v) a qu eou s solu tion of s ta rch

2.6 Det ect in g a gen t C, 10% (m /m ) h ydr och lor ic a cid


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2.7 4N h ydrochloric acid


3.1 Ch rom a togr aph y pa per (Wh at ma n pa per N o 3 an d N o 4 or t heir equ iva len ts )

3.2 Micropipet te 1l

3.3 Standard fla sks, 100ml

3.4 Fluted filters

3.5 Appa ra tus for descen ding pa per ch rom at ogr aphy


4.1 Water soluble produ cts

Make two solutions of each sample by dissolving 1g and 5g of the productrespectively in 100 ml of water. Use 1 l of each of these solutions for carrying out the

paper chroma togra phy described in Section 5.

4.2 Products sparingly soluble in water

4.2.1 We igh ou t 1 g an d 5g of t h e sa m p le a n d d is pe r se in 5 0m l of w a te r , m a k e u p t o

100ml with wa ter in each case an d mix. Filter th e two dispersions over a fluted filter

(3.4) and use 1l of each of the f i l t rates in order to carry our the paper

chromatography described in Section 5.

4 .2 .2 Prepare once again two d ispers ions of each sample by d ispers ing 1 g and 5g in 50ml

of water, acidify with dilute hydrochloric acid (2.7), make up with water to 100ml

and mix. Filter the dispersions through a fluted filter (3.4) and use 1 l of the two

filtrat es in order t o carry out th e paper chroma togra phy describedin Section 5.

4.3 Creams

Disperse 5g and 20g of each product in 100ml of water and use the dispersions to

carry out th e paper chromatography described in Section 5.

5. METHOD

5.1 P u t a n a pp rop ria t e qu a n t it y of s olve nt s A (2.2) a n d B (2 .3) in t o t wo s ep a ra t e

chromatography tanks in order to carry out the descending paper chromatography.

Sat ur at e the chr oma togra phy tan ks for at least 24 hour s with solvent vapour.

5 .2 Ap ply 1 l of on e samp le solu t ion an d of on e r efe ren ce solu t ion p rep ared accord in g to

Sections 4 and 2.1 to each start ing point on a str ip of chromatography paper

(Whatman No 3 or equivalent) 40 cm long and 20 cm wide (3.1) or another suitable

format an d evapora te th e solution in air.

5.3 P la ce t he ch r om a t ogr a ph y s tr ip (5.2) in t h e ch r om a t ogr a ph y t a n k filled wit h

developing solvent A (5.1) and develop until the solvent front has advanced 35cm

(about 15 hours).

5.4 Re pea t t h e pr oce du r e des cr ibe d in S ect ion s 5.2 a nd 5.3, us in g ch r om a t ogr a ph y p ap er

(Whatman No 4 or equivalent) (3.1) and developing solvent B. Chromatograph until

the solvent front h as advan ced 35 cm (about five hours).

5.5 Aft er developm en t rem ove t he ch rom at ogr a ms an d dr y t hem in a ir .


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5.6 Revea l t h e s pot s in t he ch r om a t ogr a m by s pr a yin g it su cces sively wit h :

5 .6.1 d et ect ing agen t A (2 .4 ) fol lowed sh or t l y t h e rea ft e r b y d et ect in g agen t B (2 .5 ). Th e

spots of the persulphates will now appear first in the chromatogram and will be

followed by the h ydrogen peroxide spots . Mark t he spots wit h a pencil;

5 .6.2 d et ect in g agen t C (2 .6 ) on th e ch romatograms ob ta in ed in accord an ce with Sect ion

5.6.1; the presence of bromates will now be indicated by greyish blue spots in the

chromatogram.

5 .7 Un der t h e ab ovemen t ion ed con d it ion s p er t a in in g to d eve lop in g solven t s A(2 .2 ) an d

B(2.3), the Rf values of th e referen ce subst an ces (2.1) ar e app roximat ely as follows:

Developing solvent

A (2.2)

Developing solvent

B (2.3)

Sodium persulphate 0,40 0,10

Potassium persulphate 0,40 0,02 + 0,05

Ammonium persulphate 0,50 0,10 + 0,20

Sodium bromate 0,40 0,20

Potassium bromate 0,40 0,10 + 0,20

Hydrogen peroxide 0,80 0,80

B. IDENTIFICATION OF BARIUM P EROXIDE

1. P RINCIP LE

Barium peroxide is identif ied by the formation of hydrogen peroxide after

acidificat ion of the sa mple (A.4.2), and by the pr esen ce of the ba riu m ion:

in the absence of persulphates (A), by adding dilute sulphuric acid to a port ion

of the acid sample solution (B.4.1), as a result of which a white precipitate of

bar ium s ulpha te is form ed. The presen ce of barium ions in th e sam ple (B.4.1) is

again confirmed by paper chromatography 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 Methanol

2.2 36% (m /m ) con cen tr at ed h ydr och lor ic a cid

2.3 6N hydroch lor ic acid

2.4 4N su lphur ic acid

2.5 Rhodizonic acid disodium salt

2.6 Bar ium ch lor ide (BaCl22H 2O)

2.7 Anhydrous sodium carbona te


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2.8 1% (m /v) a qu eou s solu tion of ba riu m ch lor ide

2.9 D eve lop in g s olve nt con s is t in g of m e th a n ol, con ce nt r a t ed h yd r och lor ic a cid

(concent ra tion 36%) and wat er (80:10:10 by vol)

2 .10 Detect ing agen t , 0 ,1 % (m/v) aqueous solu t ion of rhodizonic acid d isod ium salt , to befresh ly prepared im mediat ely before use.


3.1 Micropipet te, 5l

3.2 Plat inum crucibles

3.3 Standard fla sks, 100ml

3.4 Ch r om a t ogr a ph y p ap er S ch le ich er a n d Sch u ll 2043b or e qu iva le nt . Cle an t h e p ap er

by developing it overnight in a descending chromtography tank (A.3.5) containing

developing solvent (B.2.9) an d th en dr y.

3.5 Fluted filter paper

3.6 Th e u su al appa ra t us for ca r ryin g ou t as cen din g pa per ch rom a togr aph y


4.1 Products in which persulphate s are absent

4 .1 .1 Disper se 2g of t h e prod u ct i n 50 ml of wa ter an d br in g th e pH of t h e disper sion to

about 1 with hydr ochloric acid (B.2.3).

4.1.2 T ra n s fe r t h e d is pe rs ion wit h wa t e r in t o a 10 0-m l s t a nd a rd fla s k, m a k e u p to t h emar k with wat er a nd mix. Use this dispersion for t he paper chromatography ana lysis

described in Section 5 and for identification of barium by precipitation of the

sulphate.

4.2 Products in which persulphates are presen t

4.2.1 Dis pe rs e 2 g of t h e p rod uct in 100 ml of wa t er a n d filt er .

4 .2 .2 Add to the dr ied residue seven to 10 t imes it s weigh t of sod ium carbonate (B.2 .7), mix

an d melt t he mixtur e in a pla tinum crucible (B.3.2) for half an h our.

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 mel t in hydroch lor ic acid (B.2 .3) and make up to 100 mlwith water. Use this solution for the paper chromatography analysis described in

Section 5 an d for th e identification of barium by precipitat ion of the su lphat e.

5. METHOD

5.1 P la ce a n a pp rop r ia t e qu a n t it y of d eve lop in g s olve nt (B.2.9 ) in a t a n k for a s ce nd in g

paper chromat ography and satu rat e the ta nk for at least 15 hours.

5.2 O n a pie ce of ch r om a t ogr a p hy p a pe r p r et r ea t e d a s de scr ibe d in Se ct ion B.3.4

apply 5l of each of the solutions prepared in accordance with Sections B.4.1.2 and

B.4.2.4 an d reference solution B.2.8 at t hree s ta rt ing points.

5.3 Dr y t h e s a mp le a n d r efe re nce s pot s in a ir . D evelop t h e ch r om a t ogr a m un t il t h e

solvent front ha s as cended 30 cm.


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5.4 Rem ove t he ch rom at ogr am fr om th e t an k a nd dr y in a ir .

5.5 Revea l t h e s pot s on t h e ch r om a togr a m by sp ra yin g t h e p ap er wit h det ect in g a gen t

B.2.10. In th e presen ce of the bar ium, red spots with a n Rf value of about 0,10 appear

on the chromatogram.

C. DETERMINATION OF HYDROGEN PEROXIDE

1. P RINCIP LE

The iodometric determination of hydrogen peroxide is based on the following

reaction:

H 2O2 + 2 H+ + 2 I - I2 + 2H 2O

This conversion pr oceeds slowly but can be accelera ted by t he a ddition of amm onium

molybdate . The iod ine fo rmed i s determined t i t r imet r ical ly agains t sod iumthiosulphat e and is a m easur e of th e hydrogen peroxide cont ent.

2. DEFINITION

The hydrogen peroxide content measured in the manner described below is expressed

as a percentage by ma ss (%m/m) of the pr oduct.

3. REAGENTS


3.1 2N su lphur ic acid

3.2 Potassium iodide

3.3 Ammonium molybdate

3.4 0,1N sodium thiosulphate

3 .5 10 % (m/v) pot ass iu m iod id e solu t ion , to be fr esh ly prep ared immed ia t ely be fore u se

3.6 20% (m /v) a mm onium molybda te solut ion

3.7 1% (m/v) starch solut ion


4.1 Beakers, 100 ml

4.2 Buret te, 50ml

4.3 Standard flasks, 250 ml

4.4 Measur ing cylinders, 25 and 100 ml

4.5 One-mark pipet tes, 10ml

4.6 Conical flasks, 250 ml


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5. METHOD

5 .1 In to a 10 0-ml b eak er we igh ou t 10 g (m gram) of t h e p rod u ct , con ta in in g ab ou t 0,6 g

of hydrogen peroxide. Transfer the contents with water into a 250-ml standard flask,

make up to the mar k with water a nd mix.

5 .2 P ip et t e 1 0 ml of t h e samp le solu t ion (5 .1 ) in to a 25 0-ml con ica l fla sk (4 .6 ) an d ad d

successively 100 ml of 2N sulphuric acid (3.1), 20 ml of potassium iodide solution

(3.5) an d th ree dr ops of amm onium m olybdat e solution (3.6).

5.3 Tit r a t e t h e iod in e for m ed im m ed ia t ely a ga in s t 0,1N sod iu m th ios u lp h at e s olu t ion

(3.4) and just before the end point is reached add a few millilitres of starch solution

as indicator (3.7). Record the consumption of 0,1N sodium thiosulphate (3.4) in

millilitres (V).

5.4 I n th e ma n ne r des cr ibe d in S ect ion s 5.2 a nd 5.3, ca r ry ou t a bla n k de ter m in a tion ,

replacing th e 10ml of the s am ple solut ion by 10ml of wat er. Record th e consu mpt ion

of 0,1N sodium thiosulphat e in t he blan k det ermina tion (Vo ml).

6. CALCULATION

Calculate the hydrogen peroxide conten t of the pr oduct as a percentage by ma ss (%

m/m) with t he a id of the following form ula :

% hydrogen per oxide = (V Vo) x 1,7008 x 250 x 100

m x 10 x 1000

=(V Vo) x 4, 252

m

in which:

m = th e quan tity in gram s of product an alyzed (5.1),

Vo = the consumption in mill il it res of 0 ,1 N thiosulphate solut ion in the

blank determ inat ion (5.4),

V = th e consu mpt ion in millilitres of 0,1N th iosulpha te solut ion in th e

titration of the sample solution (5.3).


For a product containing about 6% m/m hydrogen peroxide the difference between the results of

two determinations in parallel carried out on the same sample should not exceed an absolute

valu e of 0,2%.

(1) S ee Nor m I SO 57 25 .


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II. ID EN TIF ICATION AN D S EMI-QU AN TITATIVE

DE TERMINATION OF CER TAIN OXIDATION

COLORANTS IN HAIR DYES

1. P URP OSE AND SCOP E

This method is suitable for the identification and semi-quantitative determination of

the following substa nces in h air dyes in cream or liquid form :

Substances Symbol

Phenylenediamines

o-Phenylenediamine (OPD)

m-Phenylenediamine (MPD)

p-Phenylenediamine (Annex V) (PPD)

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)

-Naphthol (-N)

Pyrogallol

1,2,3-t r ihydroxybenzene (P)

Resorcinol

1,3-dihydroxybenzene (R)

2. P RINCIP LE

Oxidation colorants are extracted at pH 10 with 96% ethanol from dyes in cream orl iqu id fo rm a nd iden t i f ied by th in-layer chr omat ograph y , e i ther one- o r

two-dimensional.

For semi-quantitative determination of these substances, the chromatogram of the

samples is compared by means of four developing systems with those for reference

substa nces produced at th e same t ime and u nder a s similar conditions a s possible.

3. REAGENTS


3.1 Ethanol, anhydrous

3.2 Acetone

3.3 Ethanol, 96% v/v


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3.4 Ammonia solu t ion 25% (d 420 =0,91)3.5 L(+)-ascorbic acid

3.6 Chloroform

3.7 Cyclohexane

3.8 Nit rogen , technical grade

3.9 Toluene

3.10 Benzene

3.11 n-Butanol

3.12 Butan-2-ol

3.13 H ypoph os ph or ou s a cid, 50% v/v solu tion

3.14 Diazo reagent . E ither :

3-Nitro-l-benzenediazonium chlorobenzenesulphonate, (stabil ized sal t form) as

in Red 2 J N Fr an color,

2-Chloro-4-n it ro-1-benzenediazonium naph tha lenebenzoat e (s ta b i lized sal t

form ) as in NN CD Reagent referen ce No 74150 FLU KA,

or an equivalent.

3.15 Silver nit r a te

3.16 p-Dimethylaminobenza ldehyde

3.17 2,5-Dimethylphenol

3.18 Ferr ic ch lor ide hexahydrate

3.19 H ydr och lor ic a cid, 10% m /v s olu tion

3.20 Reference substances

The reference substa nces are t hose shown in para graph 1 Pu rpose and scope. In the

case of amine compounds, the reference substance must be either the hydrochloride

(mono or di) or t he free base.

3.21 Refe ren ce s olu tion s 0,5% (m/v)

A 0,5% (m/v) solut ion of each of th e refere nce subs ta nces in S ection 3.20 is pr epa red.

Weigh out 50 mg 1mg of th e reference substa nce in a sta nda rd 10-ml flask.

Add 5 ml of 96% etha nol (3.3) an d 250m g of as corbic a cid (3.5).

Make the solution alkaline by addition of the ammonia solution (3.4) to give an

appa rent pH of 10 (test with indicat or pa per).

Mak e up t o 10ml with 96% eth an ol (3.3) an d mix.

The solutions m ay be kept for a week in a cool place away from th e light .

In certain cases, after the addit ion 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 ch loroform toluene (35:25:40 by vol)

3.22.2 Chloroform cyclohexane absolute ethanol 25% ammonia (80:10:10:1 by vol)


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3 .22.3 Benzene bu tan-2-ol water (50:25:25 by vol ). Shake well and use the upper phase

after sepa ra ting at r oom t empera tur e (20 to 25C).

3 .22.4 n-Butanol ch loroform reagent M (7 :70:23 by vol ). Separate carefu l ly a t room

temper at ure (20 to 25C) an d use t he lower pha se.

Preparation of reagent M

Ammonia solut ion , 25% (v/v) 24 volumes

Hypophosphorous acid, 50% (3.13) 1 volume

Water 75 volumes

Note

Developing solvents containing ammonia must be well shaken immediately 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 prepa red just before u se.

3.23.2 Ehrlichs reagent

Dissolve 2 g of p-diamethylaminobenzaldehyde (3.16) in 100ml of hydrochloric acid

10 % (m/v) aqueous solution (3.19).

3.23.3 2,5-dim ethylph enol ferric chloride hexah ydra te

S olution 1: Dissolve 1 g of dimethylphenol (3.17) in 100 ml of 96 % ethanol (3.3).

Solution 2: Dissolve 4 g of ferr ic chloride h exah ydra te (3.18) in 100 m l of 96% etha nol

(3.3).

For development, these solut ions are sprayed separately, f i rst solut ion 1 then

solution2.

3.23.4 Amm oniacal silver nitrate

25% ammonia (3.4) is added to 5% (m/v) aq. solution of silver nitrate (3.15) until the

precipitat e just dissolves. This reagent mu st be prepa red imm ediately before us e.

Do not k eep.

4. AP P ARATUS

4.1 U su al la bor at or y equ ipm en t for t hin -la yer ch rom a togr a ph y.

4.1.1 P la s t ics or g la s s cove r so con s tr u ct e d t h a t t h e ch r om a t ogr a ph ic p la t e ca n b e

surrounded with nitrogen during application of the spots and drying. This precaution

is necessa ry becaus e of the su sceptibility to oxidat ion of certa in colora nt s.

4 .1 .2 Micro-syr in ge , 1 0 l, grad u a ted in 0,2 l divi sion s , w ith a sq u are cu t n eed le , or ,

better, a 50l repeating dispenser, mounted on a clam p stan d in such a way th at the

plate can be kept under n itrogen.

4 .1 .3 S il ica ge l t h in l ayer pl a t es , r ead y to u se , 0 ,2 5 mm th i ck , formed b y 2 0 x 2 0 cm

(Macherey and Nagel, Silica G-HR, which are on plastics support, or equivalent).

4.2 Cent r ifuge, 4000 rev./min .

4.3 Cen t rifu ge tu bes , 10m l wit h PTF E -lin ed scr ew ca ps , or equ iva le nt .


44/193

Directive 82/434/EEC ___________________________________________________

38

5. P ROCEDURE

5.1 Treatmen t of tes t samples

Discar d th e first 2 or 3 cm of cream extr uded from t he tu be.

Put the following into a centrifuge tube (4.3) previously flushed out with nitrogen:

300 mg a scorbic acid with 3 g cream or 3 g homogenized liquid.

Add 25 % ammonia drop-wise (3.4) until the pH is 10. Make up to 10ml with 96%

eth an ol (3.3).

Homogenize under nitrogen (3.8), stopper and then centrifuge at 4000 rev./min. for

10 minutes.

Use the supernat ant liquid.

5.2 Chromatography

5.2.1 Spot t ing the p la tes

Under an at mosphere of nitrogen (3.8), apply to a chr oma tography plat e (4.1.3) 1l of

each of the above-described reference solutions at nine points situated about 1,5 cm

apa rt along a line appr oximately 1,5 cm from th e edge of the plat e.

These r eference solution spots a re a rr an ged as follows:

1 2 3 4 5 6 7 8 9

R P H PPD DAP PTD OPD OTD MPD

MTD -N

In addition, apply at points 10 and 11 respectively 2l of the test solution samples

obta ined a ccording t o Section 5.1.

Keep the plate u nder n itrogen (3.8) unt il the moment wh en it is chr oma tographed.

5.2.2 Development

Place the plate in a tank previously flushed out with nitrogen (3.8), saturated with

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