494/2005 Sb.
The DECREE
of 7 June. December 2005,
laying down the methods of analysis of control the composition of cosmetic products
resources
The Ministry of health provides under section 108, paragraph. 1 of law No.
258/2000 Coll., on the protection of public health and amendment to certain
related laws, as amended by Act No. 392/2005 Coll. (hereinafter referred to as
"the Act") for the implementation of section 80 (2). 2 of the Act:
§ 1
(1) this Ordinance "^ 1") incorporates the relevant provisions of the European
Community ^ 2) and modifies the following analytical methods of control
the composition of cosmetic products:
and) sampling of cosmetic products,
(b)) preparation of samples in the laboratory,
(c)) the qualitative and quantitative determination of free sodium and hydroxide
potassium,
(d)) the quantitative and qualitative determination of oxalic acid and its
alkaline salts in devices designed for hair care,
e) quantitative determination of chloroform in toothpastes,
f) quantitative determination of zinc
(g)) the qualitative and quantitative determination of
4-hydroxybenzenesulphonic,
h) qualitative determination of oxidizing agents and quantitative determination
hydrogen peroxide in devices designed in hair care,
I) qualitative and semi-quantitative determination of certain oxidation
colorants in hair dyes,
j) qualitative and quantitative determination of nitrite
to) quantitative determination of resorcinol in shampoos and hair
lotions,
l) quantitative determination of methanol in relation to ethanol or
propan-2-OL,
m) qualitative and quantitative determination of free formaldehyde,
n) quantitative determination of dichloromethane and 1, 1, 1-trichloroethane,
about) qualitative and quantitative determination of quinolin-8-OL and
bis (quinolin-8-ol)-sulfate,
p) quantitative determination of ammonia,
q) the qualitative and quantitative determination of nitromethane,
r) qualitative and quantitative determination of mercaptoacetic acid
(sulfanyloctové) in perms, straightening and
resources for hair removal,
with) the qualitative and quantitative determination of hexachlorophene (INN),
t) quantitative determination of tosylchloramide sodium (chloramine-T)
(INN)
for the quantitative determination of total) fluorine in dental creams,
in) quantitative determination of organomercury compounds,
w) quantitative determination of alkali sulphides and metal sulfides
alkaline earth metals,
x) the qualitative and quantitative determination of
2.3-dihydroxypropyl-4-aminobenzoate,
s) quantitative determination of chlorobutanol,
from) the qualitative and quantitative determination of quinine,
AA) qualitative and quantitative determination of inorganic sulphites and
hydrogen sulphites,
BB) the qualitative and quantitative determination of chlorates of the alkali
metals,
CC) the qualitative and quantitative determination of sodium iodate,
DD) the qualitative and quantitative determination of silver nitrate in
cosmetic products,
EE) qualitative and quantitative determination of the content of selenium disulphide
in anti-dandruff shampoos,
FF) quantitative determination of soluble barium and strontium in pigments
in the form of salts or complexes,
Gg) qualitative and quantitative determination of benzyl alcohol,
HH) qualitative determination of zirconium and the quantitative determination of zirconium
aluminium and chlorine in non-aerosol antiperspirants,
II) qualitative and quantitative determination of hexamidine,
dibromohexamidine, dibromopropamidine and chlorhexidine,
JJ) qualitative and quantitative determination of benzoic acid, acid
4-Hydroxybenzoic acid, sorbic acid, salicylic acid and acid
propionic acid in cosmetic products,
KK) qualitative and quantitative determination of HYDROQUINONE,
Hydroquinone-hydroquinone-monomethyletheru, monoethyletheru and
Hydroquinone-monobenzyletheru in cosmetic products,
LL) the qualitative and quantitative determination of 2-fenoxy-ethanol,
1-phenoxypropan-2-OL, methyl-, ethyl-, propyl-, butyl-and
-benzyl 4-hydroxybenzoate in cosmetic products.
(2) the contents of the analytical methods of checking composition of cosmetic products
listed in paragraph 1 is set out in the annex to this Decree.
§ 2
This Decree shall take effect on 1 January 2005. January 1, 2006.
Minister:
Mudr. Rath v. r.
Annex
1. sampling of COSMETIC PRODUCTS
1. scope and field of application
Procedure for sampling of cosmetic products is described with regard to
their analysis in different laboratories.
2. The DEFINITION of the
2.1 Basic sample:
unit quantity taken from a batch offered for sale.
2.2 the total sample:
the sum of all the core samples taken from the same lot number.
2.3. Laboratory test sample:
a representative part of the sample to be analyzed in
individual laboratories.
2.4. The test sample:
a representative part of the laboratory sample required for the analysis.
2.5 Packaging:
the object that contains the product and that the product is in constant
direct contact.
3. SAMPLING PROCEDURE
3.1
Cosmetic products must be taken in the original packaging and must be
delivered to the laboratory without having been opened.
3.2
When it comes to cosmetic products which are placed on the market in a big way
packaging or that are sold in other than the original packaging from the
the manufacturer should be issued appropriate instructions for sampling at the site
use or sale.
3.3
The number of basic samples required for the preparation of the laboratory sample
shall be determined by the analytical method and the number of analyses to be
made to each laboratory.
4. identification of
4.1
The samples at the place of sampling will ensure against opening, confusion and
contamination and identified in accordance with the rules applicable in the
the Member State concerned.
4.2.
Each basic sample taken shall bear the following information:
. the name of the cosmetic product and other available identification information
(manufacturer/importer, the event. distributor, lot)
. the date, time and place of sampling,
. the name of the person responsible for taking the sample,
. the name of the Inspectorate.
4.3.
Protocol on sampling shall be drawn up in accordance with the rules
in force in the Member State concerned.
5. STORAGE of SAMPLES
5.1
Basic samples must be stored in accordance with any instructions
the manufacturer listed on the label.
5.2.
If they are not listed other conditions, laboratory samples must be kept
in the dark place at a temperature of 10-25 ° C.
5.3.
Basic samples must not be open to analysis.
2. PREPARATION of SAMPLES in the laboratory
1. In GENERAL
1.1
If possible, it should be carried out with each analysis laboratory
sample. If the Basic sample too small, a minimum should be used
the number of basic samples. Prior to taking the test sample should be
First, thoroughly mixed together.
1.2
The packaging opens in an inert atmosphere, if required by the analytical method and the
as soon as possible to remove the required number of test samples. Then you would
the analysis should be carried out without delay. If the sample must be
retained, it should be resealed in an inert atmosphere.
1.3
Cosmetic products can be in liquid or solid form or in
semi-solid form. If the phase separation of the originally homogenous
the product should be a product sample before taking the test again
homogenized.
1.4
If the cosmetic product is put on sale in a special way, in
as a result, you cannot follow these guidelines, and if they are not
set the appropriate method of testing, custom procedure may be adopted for
provided, that will be set out in writing as part of the analysis.
2. The LIQUID
2.1
In this form there may be products, such as oil, alcohol and
aqueous solutions of lotions, toilet water, or milk, and may be packed in
flakónech, straws, ampoules or tubes.
2.2
Withdrawal of the test sample:
. before opening the container vigorously;
. the packaging is opened;
. of the corresponding amount of fluid into the tube to visually
examination of its properties for the purposes of the test sample;
. the packaging is closed again, or
. withdraw the required test samples;
. the packaging carefully.
3. SEMI-SOLIDS
3.1
In this form there may be products such as pastes, creams,
emulsions and gels and may be packed in tubes, plastic bottles or
pots.
3.2 the subscription of the test sample:
3.2.1
Containers with narrow neck:
Delete at least the first inch of the resource. Remove the test
sample and cover immediately.
3.2.2.
Wide-mouthed containers:
Scrape the top layer evenly. Remove the test portion and reseal the
immediately.
4. SOLID SUBSTANCES
4.1
In this form there may be products, such as loose powders,
Compact powders, sticks and may be packed in a wide variety of packaging.
4.2 Subscription test sample:
4.2.1.
Loose powders:
Prior to odzátkováním, or by opening the container vigorously. The packaging is
opens and removes the test sample.
4.2.2.
Compact powder or stick: scrape the top layer Evenly. From
the bottom layer with the test sample.
5. the resources in the package to AEROSOLS ("aerosol dispensers")
5.1
These products are defined in article 2 of Council Directive 75/324/EEC of
May 20, 1975 (OJ No L 147, 9. 6.1975, p. 40.)
5.2 Test the sample:
After a thorough shaking, by using the appropriate connectors (see Figure 1:
in specific cases the analytical method may be required to use
other couplings) converts a representative quantity of the contents of the aerosol
dispensers in a glass cylinder coated with a layer of plastic (Figure 4) and
fitted with an aerosol valve but without the pressure of the tube. When
convert to cylinder valve pointing down. When converting the contents well
visible. There are four options:
5.2.1.
An aerosol product in the form of a homogeneous solution, which can be directly
analyze.
5.2.2.
An aerosol product consists of two liquid phases. Each of the phases may
be analyzed after the separation of the bottom phase to the second bottle. In this case,
the first valve is facing down the bottle. In this case, is the lower stage
the aqueous and propellent (e.g. butane/water).
5.2.3.
Aerosol containing a suspension of the powder. Liquid phase can be
analyze after the separation of powder.
5.2.4
Resources in the form of a foam or cream: into the bottle with the first weigh exactly
5 to 10 g of 2-methoxyethanol. This substance prevents foaming during degassing, and
so it is possible to expel the propellent gases without loss of liquid.
5.3 Widgets
Clutch (Figure 1) is made of Duralumin or brass. Is constructed
so, in order to meet the various adapter through the polyethylene
valve systems. The picture is an example; You can use other connectors
(see figures 2 and 3).
The bottle (Figure 4) is made of white glass and from the outside is coated
a protective layer of transparent plastic. It has a volume of 50 to 100 ml.
fitted with an aerosol valve without the pressure of the tube.
5.4 the procedure
In order to transfer a sufficient amount of sample bottles must be of the
purged of air. For this purpose, to introduce through the connector about 10
ml of dichlorodifluoromethane or butane (by an aerosol device,
to be examined) and then, until the disappearance of the degassing of liquid
phase, taking the bottle with the valve pointing upward. The connector is removed. Bottle
weigh ("and" grams). Aerosol spray, which has to be removed
pattern, vigorously. The connector connects to the valve
aerosol, which has to be removed (the packaging is the valve upwards),
to the connector connects a bottle (neck down) and press it to her. The bottle is
fill about two-thirds full. If converting ceases due to settlement
pressures, refrigeration can be restored. Remove clutch, filled with
weigh the bottle ("b" grams) and determine the weight of the converted sample
aerosol m1 (m1 = b-a).
The sample thus obtained can be used:
1. for normal chemical analysis;
2. the analysis of the volatile constituents by gas chromatography.
5.4.1. Chemical analysis
With the bottle valve upwards, proceed to the following
way:
. from the Degas bottle; If foaming occurs, the
the bottle, which was introduced with a syringe through the connector in advance exactly
weighed amount of 2-Methoxyethanol (5 to 10 g);
. in a water bath at 40 ° C with shaking removes volatile components, without
There would be loss of the sample;
. consider again the bottle ("c" grams) in order to determine the weight of the rest of the m2
(m2 = c-a);
(Note: when calculating the weight of the residue, deduct the weight of any
of the quantity of 2-Methoxyethanol);
. removing the valve opens the bottle;
. the rest of the transfer in the known quantity of an appropriate
solvents;
. with an aliquot portion is done the required analysis.
Formula for calculation:
r x R x P m2
R = ------- a Q = ------ ,
M1-100
where:
M1 = mass of aerosol taken into the transfer bottle,
M2 = the mass of the residue after heating at 40 ° C,
r = the content of the particular substance in m2, expressed
as a percentage (as determined by an appropriate method),
R = the content of the individual substances in aerosol, removed
expressed as a percentage,
Q = total mass of the individual substances in aerosol
dispenser,
P = the net weight of the original the aerosol dispenser
(basic pattern).
5.4.2. The analysis of the volatile constituents by gas chromatography
5.4.2.1 the principle
Using a syringe for gas chromatography of bottle removes
a sufficient amount of sample. The contents of the syringe are injected into a gas
the chromatograph.
5.4.2.2 Aids
The exact gas-chromatography syringe series A2 on 25 mikrol up to 50
mikrol (Figure 5) or equivalent. This syringe is
equipped with a slide valve at the end of the needle. The syringe is associated with bottles
Connector on the bottle and a polyethylene tube deployed on
injection syringe (length 8 mm, internal diameter 2.5 mm).
5.4.2.3. the Procedure
After you convert a sufficient amount of an aerosol product in the bottle,
as described in item 5.4.2.2 fixed to the cylinder tapered end
the syringe. Open the valve and sucked up with a sufficient quantity of liquid.
By moving the piston removes gas bubbles (syringe
cool as needed). When the syringe is in sufficient quantity
the liquid without bubbles, close the valve and disconnect from the syringe bottle.
Deploys the needle, a syringe is inserted into the gas injector
the chromatograph, open the valve and inject.
5.4.2.4 internal standard
If using an internal standard is required, it will load into the bottle
(an ordinary glass syringe using a connector).
Figure 1
The Connector P1
Figure 2
Clutch M2
for converting between folding and objímacím valve
Figure 3
Clutch M1
for converting between the two through the valves
Figure 4
Bottle volume 50 to 100 ml
Figure 5
Syringe for gas chromatography
3. The qualitative and quantitative DETERMINATION of FREE SODIUM and HYDROXIDE
POTASSIUM
1. scope and field of application
In the method is the procedure of identification of cosmetic products
containing significant amounts of free sodium hydroxide and/or
potassium and the procedure for the quantitative determination of free sodium hydroxide
and/or potassium in straightening and paint
nail cuticle.
2. The DEFINITION of the
The amount of free sodium and potassium hydroxides is determined by the amount of
the standard volumetric solution of acid required to neutralize the device for
the prescribed conditions, and the resulting quantity shall be expressed as free
sodium hydroxide in% (m/m).
3. The PRINCIPLE of the
The sample is dissolved or disperguje in water and titrated with standard
acid solution. In parallel with adding acid records
pH value; simple solutions of sodium or potassium hydroxide is
equivalence point given by the maximum speed of the recorded values of pH changes.
The simple titration curve may be obscured by the presence of
and) ammonia and other weak organic bases, which have themselves
rather flat titration curve. In this method, the ammonia removes the
by evaporation under reduced pressure at room temperature;
b) salts of weak acids, which can cause the titration curve
has several points of inflection. In such cases corresponds to the
neutralise the hydroxyl ions originating from free
sodium or potassium only the first part of the curve to the first of these
point.
The method is given an alternative procedure for titration in alcohol cases,
when experiencing intense interference of weak inorganic salts
acids.
Although there is a theoretical possibility that a high pH may be caused by
other soluble strong principles, for example. Lithium hydroxide or
Quaternary amoniovými, hydroxides, it is their presence in these types of
cosmetic products are very unlikely.
4. QUALITATIVE DETERMINATION
4.1 reagents
4.1.1 Standard alkaline buffer, pH 9.18 at 25st. (C): 0 705 molar solution of
dekahydrátu tetraboritanu sodium.
4.2. Apparatus and equipment
4.2.1. Usual laboratory glassware
4.2.2. pH meter with a recorder
4.2.3. Glass membrane electrode
4.2.4. Standard calomel electrode referentní
4.3 Procedure
pH-meter with the electrodes shall be calibrated using the standard alkaline
the buffer solution. Prepare a 10% aqueous solution or aqueous dispersion
the product has to be analysed and filtered. Measure the pH. If the pH of the
12 or higher, must be carried out quantitative determination.
5. QUANTITATIVE DETERMINATION of
5.1. Titration in aqueous environment
5.1.1 reagent
5.1.1.1. Standard solution of hydrochloric acid 0.1 mol/l
5.1.2. Apparatus and equipment
5.1.2.1 the usual laboratory glassware
5.1.2.2. pH-meter with a recorder
5.1.2.3 Glass membrane electrode
5.1.2.4 Standard calomel electrode referentní
5.1.3 Procedure
150 ml beaker weigh accurately a test portion of the weight of 0.5
up to 1.0 g. ammonia is present add a few anti-bumping granules,
place the beaker in a vacuum desiccator, evacuate using a water
pump until the odour of ammonia (about three hours).
Add 100 ml water, dissolve or disperguje and
Titrate with 0, 1mol/l hydrochloric acid standard solution (5.1.1.1)
While recording the change in pH (5.1.2.2).
5.1.4. the calculation of the
On the titration curve inflection points are determined. If the first inflection point
It is located at a pH lower than 7 is not present in the sample free hydroxide
sodium or potassium.
If the titration curve exhibits two or more points of inflection, is for
the determination of significant, only the first of them.
Record the volume of titrant solution to achieve the first inflection
point.
If the titrant, in ml and M is the mass of the test sample in the
grams, calculate the content of sodium and/or potassium hydroxides in the sample
expressed as sodium hydroxide in% (m/m), using the formula
In
% = 0,4 ---
M
The situation may arise when a significant presence through indications
the quantity of sodium and/or potassium hydroxides shows titration curve
a clear inflection point. In this case, you must repeat the determination in
isopropyl alcohol.
5.2. Titration in isopropyl alcohol
5.2.1 Used chemicals
5.2.1.1. Isopropyl alcohol
5.2.1.2 Aqueous hydrochloric acid, 1.0 mol/l
5.2.1.3 0.1 mol/l hydrochloric acid solution in isopropyl alcohol
prepared immediately before use by diluting the 1.0 mol/l aqueous
hydrochloric acid isopropyl alcohol
5.2.2 apparatus
5.2.2.1. Usual laboratory glassware
5.2.2.2. pH-meter
5.2.2.3. Glass membrane electrode
5.2.2.4. Standard calomel electrode referentní
5.2.3 Procedure
150 ml beaker weigh accurately a test portion of the weight of 0.5
up to 1.0 g. ammonia is present add a few anti-bumping granules,
place the beaker in a vacuum desiccator, evacuate using a water
pump until the odour of ammonia (about three hours).
Add 100 ml of isopropyl alcohol, the rest in a beaker, dissolve or
disperguje and Titrate with the 0.1 mol/l standard solution of acid
hydrochloric acid in isopropyl alcohol (5.2.1.3) recording
change of pH (5.2.2.2).
5.2.4 Calculation
As in section 5.1.4. The first inflection point lies approximately at the measured values
pH 9.
5.3 the repeatability (see ČSN ISO 5725)
For products containing about 5% (m/m) of sodium or potassium hydroxide,
expressed as sodium hydroxide, the difference between the results of two
parallel on the same sample should not exceed the determination of the absolute value of the
0.25%.
4. the quantitative and QUALITATIVE DETERMINATION of OXALIC ACID and its
ALKALINE SALTS IN DEVICES DESIGNED FOR HAIR CARE
1. scope and field of application
The method described below is suitable for quantitative and qualitative
determination of oxalic acid and alkaline salts in
for hair care. It can be used for colourless aqueous or alcoholic
solutions and lotions which contain about 5% oxalic acid or
the equivalent amount of alkaline oxalate.
2. The DEFINITION of the
Content of oxalic acid and/or its alkaline salts determined by this
method is expressed in percentage by mass (m/m) of the free acid
oxalic acid in the sample.
3. The PRINCIPLE of the
After the removal of all present anionic surfactants
using p-toluidinu hydrochloride with oxalic acid and/or alkaline
oxalates precipitated as calcium oxalate and filter the solution.
The precipitate is dissolved in sulphuric acid and Titrate with permanganate
potassium.
4. reagents
All the reagents must be of analytical purity or higher
4.1. Ammonium acetate solution, 5% (m/m)
4.2 calcium chloride solution, 10% (m/m)
4.3. Ethanol, 95% (V/V)
4.4. Carbon tetrachloride
4.5. diethyl ether
4.6. Solution of p-toluidine hydrochloride, 6.8% (m/m)
4.7. Potassium permanganate solution, 0.5 mol/l
4.8. sulphuric acid, 20% (m/m)
4.9. hydrochloric acid, 10% (m/m)
4.10. sodium acetate trihydrate,
4.11. Glacial acetic acid
4.12. sulphuric acid (1:1)
4.13. Saturated barium hydroxide solution
5. apparatus and EQUIPMENT
5.1. separating funnels, 500 ml
5.2. Beakers, 50 ml and 600
5.3 the crucibles of G4
5.4. Measuring cylinders, 25 and 100 ml
5.5. Pipettes, 10 ml
5.6 Pump at 500 ml
3.5 Water pump
5.8. Thermometer graduated from 0 to 100 ° C
5.9. Magnetic stirrer with hot plate
5.10 insert the Teflon coated Magnetic
5.11 Burette, 25 ml
5.12. conical flasks, 250 ml
6. the procedure
6.1
50-ml beaker weigh out 6 to 7 grams exactly the sample, pH is adjusted to
the value 3 with dilute hydrochloric acid (4.9), and transfer the solution
into the separating funnel, 100 ml of distilled water. Gradually add 25
ml of ethanol (4.3), 25 ml of p-toluidine hydrochloride (4.6) and 25
up to 30 ml of carbon tetrachloride (4.4) and shake violently.
6.2.
After phase separation, the lower (organic) layer, the extraction
repeated after adding the reagents mentioned in 6.1 and organic
the layer again to forgive.
6.3.
The aqueous layer into a beaker, 600 ml on and any
Carbon tetrachloride is removed by boiling the solution.
6.4.
Add 50 ml of ammonium acetate solution (4.1), bring the solution to the boil
(5.9) and stir into the boiling 10 ml of hot chloride solution
calcium (4.2); the precipitate settle.
6.5.
The completeness of the precipitation shall be verified by adding a few drops of chloride solution
calcium (4.2), allow to cool to room temperature and
then stir in 200 ml of ethanol (4.3); (5.10); leave for 30 minutes
State.
4.1
The supernatant liquid through a glass filter Crucible (5.3),
transfer the precipitate in the filter Crucible using a small amount of
hot water (50 to 60 ° C) and wash with cold water.
4.2
Wash the precipitate five times with a small amount of ethanol (4.3), five
small quantities of ethyl ether (4.5) and dissolve in 50 ml of hot
sulphuric acid (4.8), which lets through a filter crucible for
the vacuum.
4.2
Quantitatively transfer the solution into a conical flask (5.12) and Titrate
potassium permanganate solution (4.7) to the faintly pink coloring.
7. The CALCULATION of the
Content of oxalic acid in the sample, expressed as percentage by weight
is calculated from the formula
And x 4.50179 x 100
oxalic acid content in% =--------------------
(E) x 1000
where
A = consumption 0.5 mol/l potassium permanganate solution as specified in point
4.2,
E = portion of the sample in grams (6.1),
4.50179 = conversion factor for oxalic acid.
8. REPEATABILITY
For products containing about 5% (m/m) the difference between the oxalic acid
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.15%.
9. QUALITATIVE DETERMINATION
9.1 the principle
Oxalic acid and oxalates are precipitated or as calcium oxalate
and dissolved in sulphuric acid. The solution adds a small amount of the solution
of potassium permanganate, which loses its colour for the formation of carbon dioxide.
If the resulting carbon dioxide, barium hydroxide solution, creates a
the white precipitate (milkiness) of barium carbonate.
9.2 Procedure
9.2.1.
Part of the sample to be analyzed, shall be subjected to the procedure described in
6.1 to 6.3; will, where appropriate, present detergents.
9.2.2
To approximately 10 ml of the solution referred to in point 9.2.1 Add a spatula tipful
sodium acetate (4.10) and acidify a few drops ice
acetic acid (4.11).
9.2.3.
Add 10% calcium chloride solution (4.2) and filter the solution.
Dissolve the calcium oxalate precipitate in 2 ml of sulphuric acid
(1:1) (4.12).
9.2.4
Transfer the solution into a test tube and drop add about 0.5 ml of 0.5
mol/l potassium permanganate solution (4.7). In the presence of oxalate
the solution loses its colour, first slowly and then quickly.
9.2.5
Immediately after adding the potassium permanganate solution the tube closes
stopper the tube, heat the contents slightly and the resulting carbon dioxide
in a saturated barium hydroxide solution (4.13). The formation of milk
cataracts of barium carbonate during 3 to 5 minutes, indicates the presence of
oxalic acid.
5. QUANTITATIVE DETERMINATION of CHLOROFORM in toothpaste
1. scope and field of application
Method is used for the quantitative determination of chloroform in toothpaste
by gas chromatography. It is suitable for the determination of chloroform on the
concentration of 5% or lower. Currently, the prohibition of the use
chloroform in cosmetics.
2. The DEFINITION of the
The chloroform content of the sample determined by this method is expressed in
percentage by weight of the product weight.
3. The PRINCIPLE of the
Toothpaste is suspended in a dimethylformamide/methanol, to which
It adds a known quantity of Acetonitrile as internal standard. After
spin is part of the liquid phase analyses by gas chromatography and
calculate the chloroform content.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Porapak Q, Chromosorb 101 or equivalent 80 to 100 mesh
4.2 Acetonitrile
4.3 Chloroform
4.4 Dimethylformamide
4.5 Methanol
4.6. Internal standard solution
5 ml of dimethylformamide (4.4) pipette into the flask, 50 ml,
add about 300 mg of Acetonitrile (M mg), make up to the mark with
dimethylformamide and mix.
4.7 the Solution to determine the relative response factor
Exactly 5 ml of the internal standard solution (4.6) into a volumetric pipette
10 ml flask and add about 300 mg of chloroform (M1 mg). Make up
with dimethylformamide and mix.
5. apparatus and EQUIPMENT
5.1. analytical balance
5.2. Gas Chromatograph, with flame ionization detector
5.3 micro 5 to 10 mikrol, graduated in 0.1 mikrol
5.4. bulb pipettes for 1, 4 and 5 ml
5.5 volumetric flasks, 10 and 50 ml
5.6
Test tubes, approximately 20 ml, with screw caps, Sovirel France No 20 or
equivalent. The screw cap has an internal sealing surface-coated
Teflon.
5.7
Centrifuge
6. the procedure
6.1 Featured chromatographic conditions
6.1.1.
Column material: glass
length: 150 cm
internal diameter: 4 mm
external diameter: 6 mm
6.1.2.
The column is filled with Porapakem Q, Chromosorb 101 or equivalent
filling, 80 to 100 mesh (4.1).
6.1.3
Flame ionization detector: sensitivity is set so that when the
injection 3 mikrol solution 4.7 was the height of the acetonitrile peak about three
a quarter of the entire range of recorders.
6.1.4.
Gases:
Carrier gas: nitrogen, flow rate 65 ml/min.
Auxiliary gas: hydrogen, air or oxygen
The flow of gases to the detector so that the flow of air or
oxygen was a five-to 10 times the flow rate of hydrogen.
6.1.5
Temperature:
the injection of 210 ° C
210 ° C detector
column 175 ° C
6.1.6
Chart speed:
about 100 cm per hour.
6.2. Preparation of the sample
The sample for analysis shall be taken from an unopened tube. Remove one
a third of the content, the tuba is closed, the contents are thoroughly and remove the
the test sample.
6.3. The quantitative determination of
6.3.1.
In a test tube with screw cap (3.5), weigh to the nearest 10 mg
6 to 7 grams of toothpaste (M0 grams) according to point 6.2 and add three
small glass balls.
6.3.2.
In test tubes, pipette exactly 5 ml of the internal standard solution
(4.6), 4 ml of dimethylformamide (4.4) and 1 ml of methanol (4.5) and place the
close and mix.
6.3.3.
Closed tube inserts for half an hour into the Shaker and then 15 minutes
centrifuge at the speed at which there is a clear separation of the phases.
Note: it occasionally happens that after centrifugation of the liquid phase is still
cloudy. A certain improvement can be achieved by adding 1 to 2 grams
sodium chloride to the liquid phase and recentrifuging.
6.3.4.
3 mikrol of this solution (6.3.3) under the conditions described in the inject
6.1. The procedure is repeated. Under the above conditions can be
the retention times of the Guide:
methanol of approximately 1 min.,
acetonitrile approximately 2.5 min.,
chloroform approximately 6 min.,
dimethylformamide > 15 min.
6.3.5.
Determination of relative response factor
For the determination of this factor inject 3 mikrol solution as specified in point
4.7. the procedure is repeated. The relative response factor is determined each day.
7. The CALCULATION of the
7.1. Calculation of the relative response
7.1.1.
Measure the height of the acetonitrile and chloroform peaks and their width, in
half height and their area is calculated by the formula: height × width in
half height.
7.1.2. Determine the area of the acetonitrile and chloroform peaks in the chromatogram
recorded in accordance with section 6.3.5 and calculate the relative response
FS from the following formula:
As. As Me. 1/10 M
fs = ----------- = --------------
Ms. Ai Ai. M1
where
FS = the relative response factor for chloroform,
As = the area of the chloroform peak (6.3.5);
AI = the acetonitrile peak (6.3.5);
Ms = the quantity of chloroform in mg per 10 ml of the solution according to the
section 6.3.5 (= M1)
MI = the quantity of Acetonitrile in mg per 10 ml of the solution according to the
section 6.3.5 (= 1/10 m).
Calculate the mean of the measured values.
7.2 calculation of the chloroform content
7.2.1.
Procedure referred to in point 7.1.1, calculate the area of the chloroform and
acetonitrile in the chromatogram of the recorded procedure referred to in point
6.3.4.
7.2.2.
The chloroform content in the toothpaste is calculated using the following formula:
As. As Me. M
%X = --------------- . 100% = ----------------------
FS. The MSX. AI fs. AI. Mo. 100
where
% X = the chloroform content in the toothpaste expressed by mass
as a percentage,
As = the area of the chloroform peak (6.3.4)
AI = the acetonitrile peak (6.3.4)
MSX = weight of the sample referred to in section 6.3.1 of mg (= 1000 ˇ M0)
MI = the quantity of Acetonitrile in mg per 10 ml of the solution according to the
section 6.3.2 (= 1/10 m).
Calculate the mean of the levels found and express the result to the nearest
at 0.1%.
8. REPEATABILITY
For products containing about 3% (m/m) of chloroform, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.3%.
6. QUANTITATIVE DETERMINATION of ZINC
1. scope and field of application
Method is suitable for the determination of zinc present in cosmetic
resources in the form of chloride, sulphate or 4hydroxybenzensulfonátu
zinc oxide, or as a combination of these salts.
2. The DEFINITION of the
The zinc content of the sample determined gravimetrically as the bis (2methyl-
Zinc 8oxochinolinát) and expressed in percentage by mass of zinc
in the sample.
3. The PRINCIPLE of the
Zinc present in the solution is precipitated in an acid medium such as bis (2-
Zinc methyl8oxochinolinát). After filtration, the precipitate is dried and
will consider.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1
Concentrated ammonia, 25% (m/m); d204 = 0.91
4.2.
Glacial acetic acid
4.3.
Ammonium acetate
4.4.
2-methylquinolin-8-ol
4.5.
Ammonia solution, 6% (m/V)
240 g of concentrated ammonia solution (4.1) into a volumetric
1000 ml flask, make up to the mark with distilled water and mix thoroughly.
4.6 ammonium acetate solution, 0.2 mol/l
15.4 g of ammonium acetate (4.3) in distilled water, in a
1000 ml flask, make up to volume and mix thoroughly.
4.7 the Solution 2-methylquinolin-8-OL
5 g 2-methylquinolin-8-OL in 12 ml of glacial acetic acid,
transfer to a volumetric flask with distilled water to 100 ml. Make up
mark with distilled water and mix thoroughly.
5. apparatus and EQUIPMENT
5.1. volumetric flasks, 100 and 1000 ml
5.2. Beakers, 400 ml of the
5.3. Measuring cylinders, 50 and 150 ml
5.4. Graduated pipettes, 10 ml
5.5 crucibles G-4
5.6 Pump at 500 ml
3.5 Water pump
5.8. Thermometer graduated from 0 to 100 ° C
5.9. Desiccator with Silicagel and humidity indicator,
for example. is silica gel or an equivalent means of drying
5.10 drying oven regulated to a temperature 150 +/-2 ° C
5.11 pH meter
5.12 the heating plate
5.13. Filter paper, Whatman No 4 or equivalent
6. the procedure
1.400 ml beaker, 5 to 10 g (M gram) of exactly
the analysis of the sample, which contains about 50 to 100 mg of zinc, add
50 ml of distilled water and mix thoroughly.
1. filter the mixture as needed by using the vacuum pump and the filtrate.
2. Repeat the extraction step with the other 90 ml of distilled water. Filter and
filtrates.
6.2.
For every 10 mg of zinc present in the solution (6.1) add 2 ml
2methylchinolin8olu solution (4.7) and mix.
6.3.
Dilute the mixture with 150 ml of distilled water, heated to 60 ° C (5.12)
stirring constantly, add 45 ml of solution, 0.2 mol/l ammonium acetate (4.6).
6.4.
the pH of the solution is adjusted to the stirring 5.7 to 5.9 by adding 6%
ammonia solution (4.5); the pH of the solution is measured by the pH meter.
6.5.
The solution to stand for 30 minutes. Then filter using a water pump
through a G-4 filter Crucible, previously dried (150 ° C) and cool
weighed (M0 grams); wash the precipitate with 150 ml of distilled water heated to
at 95 ° C.
4.1
The Crucible is placed in an oven at 150 ° C and the dry one
an hour.
4.2
Crucible from the oven, place in a desiccator (5.9) and after
It has cooled down to ambient temperature, weigh (M1 grams).
7. The CALCULATION of the
The zinc content of the sample, in percentage by mass (% m/m)
calculated using the formula:
(M1-MO) × 17.12
% zinku = --------------------
M
where
M = mass of the sample taken in accordance with point 6.1 (g),
M0 = the mass of the empty dry filter Crucible (6.5)
in (g),
M1 = mass of the precipitate in the filter Crucible (4.2) in (g).
8. REPEATABILITY
For products that contain about 1% (m/m) the difference between the results of zinc
two parallel on the same sample should not exceed the determination of absolute
the value of 0.1%.
7. The qualitative and quantitative DETERMINATION of 4-
HYDROXYBENZENESULPHONIC
1. scope and field of application
Method is suitable for the identification and determination of phenolsulfonic acid
in cosmetics, for example. in aerosols and lotions
lotions.
2. The DEFINITION of the
4-hydroxybenzenesulphonic acid content in the product, as determined by the
method is expressed in percentage by weight of anhydrous-4
hydroxybenzenesulfonate zinc.
3. The PRINCIPLE of the
The test sample is concentrated under reduced pressure, dissolved in water and
purified by extraction with chloroform. 4hydroxybenzensulfonová acid,
be determined iodometrically in aliquot the filtered aqueous solution.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Concentrated hydrochloric acid, 36% (m/m) (d204 = 1.18)
4.2 Chloroform
4.3 the Butan-1-ol
4.4. Glacial acetic acid
4.5. potassium iodide
4.6. Potassium bromide
4.7. sodium carbonate
4.8. Sulphanilic acid
4.9. Sodium nitrite
4.10 the potassium bromate, 0.6 mol/l
4.11. Sodium Thiosulphate solution, 0.2 mol/l
4.12 Aqueous starch solution, 1% (m/V)
4.13. sodium carbonate aqueous solution, 2% (m/V)
4.14 the aqueous solution of sodium nitrite, 4.5% (m/V)
4.15 the dithizone in chloroform Solution, 0.05% (m/V)
4.16
Mobile phase: butan-1-ol/glacial acetic acid/water (4:1: 5 V/V/V);
After mixing in the separating funnel to the bottom layer.
4.17. Pauly reagent
4.5 g of sulphanilic acid (4.8) in 45 ml of concentrated
hydrochloric acid (4.1) and dilute with water to the
500 ml 10 ml of the solution is cooled in a bowl with ice water and stir
Add 10 ml of cold sodium nitrite solution (4.14). The solution is
stand for 15 minutes at 0 ° C (at this temperature the solution is stable
After one to three days) and immediately before spraying (7.5) add 20
ml of sodium carbonate solution (4.13).
4.18 Finished cellulose plates for thin-layer chromatography;
size 20 x 20 cm, layer thickness 0.25 mm sorbent.
5. apparatus and EQUIPMENT
5.1 flask 100 ml round-bottom and ground glass stopper
5.2. Separating funnel, 100 ml
5.3 Erlenmeyer flask with ground-glass joint 250 ml
5.4 a 25 ml burette
5.5. bulb pipettes, 1, 2 and 10 ml
5.6 Single pipette, 5 ml
5.7 the microsyringe of 10 mikrol graduated in 0.1 mikrol
5.8. Thermometer graduated from 0 to 100 ° C
3.7. water bath with heating
5.10 drying oven, well ventilated and regulated at a temperature of 80 ° C
5.11 the Normal equipment for thin-layer chromatography
6. PREPARATION of the SAMPLE
In the method described below for qualitative and quantitative determination of
hydroxybenzenesulphonic acid in aerosols can be used the rest of the
obtained after the aerosol containers of solvents and driving
gases, which evaporate at normal pressure.
7. QUALITATIVE DETERMINATION
7.1
By using a micro-syringe (5.7) apply 5 mikrol residue (6) or sample
the starting line within six points at a distance of 1 cm from the lower edge of the
plates for thin-layer chromatography (4.18).
7.2.
Place the plate in a tank containing mobile phase (4.16) and
develop until the solvent front has reached 15 cm from the starting line.
7.3.
The plate from the bath and dry at 80 ° C, until the smell of
acetic acid. Spray the plate with sodium carbonate solution (4.13), and
dry on air.
7.4.
One half of the plate with a glass plate and cover the uncovered part of the
0.05% dithizone solution spray solution (4.15). The emergence of purplish red
spots in the chromatogram indicates the presence of zinc ions.
7.5
Spattered part covers the glass plate and the remainder of the
"Mihail osipovitch. Pauly reagent (4.17). The presence of 4-
hydroxybenzenesulphonic is indicated by the brown spots with
an Rf value of about 0.26, while a yellow spot with an Rf value of about 0.45 to
the chromatogram indicates the presence of 3-hydroxybenzenesulphonic acid.
8. The QUANTITATIVE DETERMINATION of
8.1.
The flask 100 ml round-bottomed, weigh out 10 g of the sample or residue (6)
and evaporate almost to dryness under vacuum in a rotary evaporator placed on the water
bath kept at 40 ° C.
8.2.
The pipette 10.0 ml bottles of water (V1 ml) and dissolve the evaporation residue (8.1) by heating
dissolve.
8.3
Quantitatively transfer the solution into a separating funnel (5.2) and extract
twice 20 ml of chloroform (4.2). After each extraction, the
the chloroform layer.
5.2
Filter the aqueous solution through a fluted filter. According to the expected content
hydroxybenzenesulphonic acid 1.0 or 2.0 ml to (V2) of the filtrate
into the 250 ml conical flask (5.3) and dilute to 75 ml
water.
5.3
Add 2.5 ml of 36% hydrochloric acid (4.1) and 2.5 g of bromide
potassium (4.6), mix and bring the temperature of the water bath at 50th.
(C).
5.3
From a burette, add 0.6 mol/l solution of potassium bromate (4.10),
until the solution at 50 ° C turns yellow.
5.4
Add 3 ml of potassium bromate solution (4.10), stopper the
and allow to stand for 10 minutes in a water bath at 50 ° C.
If, within 10 minutes the solution loses its colour, add another 2.0 ml
of potassium bromate solution (4.10), stopper the flask and heat for 10
minutes in a water bath at 50 ° C shall be the total quantity.
potassium bromate solution added (and).
5.5
Cool the solution to room temperature add 2 g of iodine
potassium (4.5) and mix.
5.5
The iodine liberated is titrated 0.2 mol/l sodium Thiosulphate solution (4.11).
At the end of the titration add a few drops of starch solution (4.12) as
indicator. Record the volume of Thiosulphate solution required, (b).
9. The CALCULATION of the
The contents of hydroxybenzensulfonátu zinc oxide in the sample or residue (6)
expressed in percentage by mass (% m/m) is calculated from the formula:
(% m/m) of hydroxybenzensulfonátu zinc oxide
(a-b) x V1 x 0.00514 x 100
= --------------------------------
m x V2
where
a = the total quantity added 0.6 mol/l solution of bromate
(5.4) in ml,
b = the total amount of 0.2 mol/l sodium Thiosulphate solution
consumed in the reverse titration (8.9),
m = the quantity of the sample or residue (8.1) in mg,
V1 = the volume of the solution obtained in item (5.1) in millilitres,
V2 = the volume of dissolved the rest after evaporation, used for the analysis
(5.2) in millilitres.
Note: when the aerosols must be the result of the determination in
percentage by weight,% (m/m) of the residue (6) converted into the original
product. For this purpose, see the rules of collection of aerosols.
10. REPEATABILITY
For products containing about 5% (m/m) of hydroxybenzensulfonátu zinc oxide
the difference between the results of two parallel determinations on the same sample
shall not exceed the absolute value of 0.5%.
11. INTERPRETATION of RESULTS
According to the Decree of the Ministry of health no. 26/2001 Coll., on the
hygiene requirements for cosmetic products, on the terms of
the request for the omission of the ingredients on the packaging of cosmetic product and the
requirements for education and experience of the natural person responsible for the production of
a cosmetic product and the assessment of the safety of a cosmetic product
for the health of individuals (cosmetic products) is the highest
the permitted concentration of zinc in the skin 4hydroxybenzensulfonátu
lotions and deodorants to 6% (m/m). This means that in addition to the content
hydroxybenzenesulphonic acid must be determined and the zinc content.
By multiplying the calculated hydroxybenzensulfonátu zinc oxide content (9)
factor 0.1588 minimum zinc content in% (m/m) that must
theoretically be present in the product with regard to fixed content
hydroxybenzenesulphonic acid. Real, by fixed
the zinc content (see the relevant provisions) may, however, be higher as
cosmetic products may also contain zinc chloride and sulfate
zinc.
8. qualitative DETERMINATION of the OXIDIZING AGENTS and QUANTITATIVE DETERMINATION
HYDROGEN PEROXIDE IN DEVICES DESIGNED FOR HAIR CARE
SCOPE AND FIELD OF APPLICATION
Iodometric determination of hydrogen peroxide in cosmetics is only possible for
the absence of other oxidizing agents, producing iodine from iodides. From
for this reason the iodometric determination of hydrogen peroxide necessary to
to detect and identify any other oxidizing agents present. This
qualitative determination shall be divided into two phases; the first includes the
peroxodisírany, bromates and hydrogen peroxide, the latter barium peroxide.
AND QUALITATIVE DETERMINATION OF PEROXODISÍRANŮ, BROMATES AND HYDROGEN PEROXIDE
1. The PRINCIPLE of the
Peroxodisíran sodium, potassium and ammonium peroxodisíran peroxodisíran,
potassium bromate, sodium bromate and hydrogen peroxide-regardless of
whether it comes or does not come from barium peroxide-identify
using descending paper chromatography, which uses two
Mobile phases.
2. USED CHEMICALS
All the reagents must be of analytical purity or higher
2.1 the 0.5% (m/V) aqueous reference solutions of the following compounds:
2.1.1 Peroxodisíran disodium
2.1.2 Peroxodisíran didraselný
2.1.3 Peroxodisíran diamonný
2.1.4 potassium Bromate
2.1.5 sodium Bromate
2.1.6 hydrogen peroxide
2.2 Mobile phase, 80% (V/V) ethanol
2.3 mobile phase B, benzene-methanol-water 3methylbutan1ol-(34:38
: 18:10 V/V/V/V)
2.4 Detection reagent A, 10% (m/V) aqueous solution of potassium iodide
2.5 the detection reagent B, 1% (m/V) aqueous solution of starch
2.6 Detection agent C, 10% (m/m) hydrochloric acid
2.7 4 mol/l hydrochloric acid
3. apparatus and EQUIPMENT
chromatography paper (Whatman paper No. 3 or 4 or their
equivalents)
3.2 the Micropipette to 1 mikrol
3.3. volumetric flasks, 100 ml
3.4 folded filters
3.5 Device to the downward paper chromatography
4. PREPARATION of the SAMPLE
4.1 Means soluble in water
From each sample prepare two solutions by dissolving 1 g and 5 g
resource in 100 ml of water. 1 mikrol each of these solutions will be used
for the implementation of the paper chromatography described in section 5.
4.2 Resources partly soluble in water
Weigh 1 g and 5 g of the sample and disperse in 50 ml of water, in both cases
make up to 100 ml with water and mix thoroughly with both suspensions to disposal
through a fluted filter (3.4) and of each of the filtrates 1 mikrol
for the implementation of the paper chromatography described in section 5.
4.2.2.
Once again, prepare two suspensions of samples of distraction 1 g and 5 g 50
ml water, acidify with dilute hydrochloric acid (2.7)
water to 100 ml and mix. Dispersion is through the stacked
filter (3.4) and 1 mikrol each of these solutions will be used for
implementation of the paper chromatography described in section 5.
4.3 Creams
5 g and 20 g of each resource is dispersed in 100 ml of water and dispersion is
apply for the implementation of the paper chromatography described in section 5.
5. the procedure
5.1
A reasonable quantity of solvents A (2.2) and B (2.3) into two
separated by chromatographic Chambers, to falling paper
chromatography. Saturate the chromatography tank with the vapours of solvents
at least 24 hours.
5.2.
1 mikrol sample solution and all reference solutions prepared in accordance with
section 4 and section 2.1 is spotted at the starting points of the banner
chromatography paper (Whatman No 3 or equivalent) 40 cm
long and 20 cm wide (3.1) or other relevant measures, and
allow the solvent to evaporate in the air.
5.3.
Chromatography Strip (5.2) is placed in the chromatography tank
tank filled with developing solvent A (3.2) and develop until the forehead
the solvent shall not refer to about 35 cm (about 15 hours).
5.4.
The procedure described in paragraphs 5.2 and 5.3 shall be carried out as well using the
chromatography paper (Whatman No 4 or equivalent) (3.1) and
(B) mobile phase chromatography is performed until the solvent front
shall not refer to about 35 cm (about five hours).
5.5
After developing the chromatograms are removed and dried air.
5.6 the Spots appear after then splashes:
5.6.1
detection reagent A and reagent B shortly after detection (2.5). The spots
peroxodisíranů on the chromatogram appears as soon as possible and will
follow spots of hydrogen peroxide. Patches should be marked with a pencil;
5.6.3
detection reagent C (2.6) in the chromatograms obtained in accordance with section
5.6.1; the presence of bromates greyish blue spots are reflected on
the chromatogram.
7. Under the abovementioned conditions pertaining to developing solvents
And (2.2) and B (2.3) are the Rf values of the reference substances (2.1)
the following:
--------------------------------------------------------------
The mobile phase and (2.2) of the mobile phase B (2.3)
--------------------------------------------------------------
Peroxodisíran 0.40 0.10
sodium
--------------------------------------------------------------
Peroxodisíran 0.40 0.02 + 0.05
potassium
--------------------------------------------------------------
Peroxodisíran 0.50 + 0.10 0.20
ammonium
--------------------------------------------------------------
Sodium bromate 0.40 0.20
--------------------------------------------------------------
Potassium bromate 0.40 + 0.10 0.20
--------------------------------------------------------------
Hydrogen peroxide 0.80 0.80
--------------------------------------------------------------
(B). QUALITATIVE DETERMINATION OF BARIUM PEROXIDE
1. The PRINCIPLE of the
Barium peroxide is identifies, on the basis of making hydrogen peroxide after
acidification of the sample (4.2) and on the basis of the presence of barium ions:
. in the absence of peroxodisíranů (A) by the addition of dilute sulphuric acid
in the part of the acid sample solution (b.4.1), to produce a white precipitate
barium sulphate. The presence of barium ions in the sample (b.4.1) is again
confirm paper chromatography in the manner described below (b.5),
. in the samples in which barium peroxide and peroxodisírany
present at the same time (B. 4.2), digesting the residue from the solution (4.2) in the
alkaline environment; After the dissolution of the melt (B. 4.2.3) in acid
the presence of barium ions hydrochloric confirm paper
chromatography and/or by precipitation of barium sulphate.
2. reagents
2.1 Methanol
2.2 concentrated hydrochloric acid, 36% (m/m)
2.3. hydrochloric acid, 6 mol/l
2.4. sulphuric acid, 8 mol/l
2.5 rhodizonové acid disodium salt
2.6 barium chloride (Bacl2 2H2O)
2.7. Anhydrous sodium carbonate
2.8. Aqueous solution of barium chloride, 1% (m/V)
2.9. Mobile phase, methanol-concentrated hydrochloric acid
(concentration 36%)/water (80:10:10 V/V/V)
2.10 the detection agent, 0.1% (m/V) aqueous solution of disodium salts of
rhodizonové, prepare immediately before use
3. apparatus and EQUIPMENT
3.1 the Micropipette to 5 mikrol
3.2 Platinum crucibles
3.3. volumetric flasks, 100 ml
3.4. Chromatography paper Schleicher and Schull 2043 b or
equivalent.
The paper up so that overnight develop in the Chamber for
descending chromatography (3.5), containing mobile phase, and then
dry.
3.5 the pleated filter paper
3.6 Normal equipment for carrying out ascending paper chromatography
4. PREPARATION of the SAMPLE
devices which are not present peroxodisírany
4.1.1
2 g of the product in 50 ml of water and the pH of the suspension is adjusted
approximately 1 hydrochloric acid (b.2.3).
4.1.2
Suspension is by using water on a volumetric flask, make up to 100 ml
volume with water and mix. This suspension shall be used for the analysis
the paper chromatography described in section 5 and for the qualitative
determination of barium by precipitation of the sulphate.
4.2 Resources, which are present in peroxodisírany
4.2.1.
2 (g) of the resource are dispersed in 100 ml of water and filter.
4.2.2.
To vysušenému the rest of the amount of sodium carbonate are added corresponding
seven to 10 times the weight of the residue (2.7), mix and blend with the
half an hour to melt in a Platinum Crucible (b.3.2).
4.2.3.
Cool to room temperature, melt, dissolve in 50 ml of water and
filter (B. 3.5).
4.2.4
The rest of the melt, dissolve in hydrochloric acid (b.2.3) and
make up with water to 100 ml. This solution is used for the analysis
the paper chromatography described in section 5 and for the qualitative
determination of barium by precipitation of the sulphate.
5. the procedure
5.1
A reasonable quantity of mobile phase (2.9) is placed in the Chamber
bullish chromatography and saturate the Chamber with at least 15 hours.
5.2.
On the chromatography paper-in advance of the adjusted as described in the
point (B). 3.4-apply to the three starting points of each of the 5 mikrol
the solutions prepared in accordance with points (B) and (B). 4.1.2.4.2.4 and reference solution
(B) 2.8.
5.3.
The sample and reference spots is dried in the air. Chromatogram
develops, until the solvent front has advanced about 30 cm.
5.4.
Chromatogram of the plate from the tank and dry in air.
5.5
Spots on the chromatogram appears after spraying the paper detection
reagent B 2.10. In the presence of the barium, red appear on the chromatogram
spots with an Rf value of about 0.10.
(C). THE QUANTITATIVE DETERMINATION OF HYDROGEN PEROXIDE
1. The PRINCIPLE of the
Iodometric determination of hydrogen peroxide is based on the following
response:
H2O2 + 2 h + + 2I-> I2 + H2O
This conversion occurs slowly, but it can be accelerated by adding
ammonium molybdate. The iodine formed is determined titračně thiosulfate
is the measure of the content of sodium and hydrogen peroxide.
2. The DEFINITION of the
The hydrogen peroxide content measured in the manner described below is expressed in
percentage by mass (% m/m).
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 the sulphuric acid 4 mol/l
3.2. potassium iodide
3.3. Ammonium molybdate
3.4. Sodium Thiosulphate, 0.2 mol/l
3.5. Potassium Iodide solution, 10% (m/V),
prepare immediately before use
3.6. ammonium molybdate Solution, 20% (m/V)
3.7. Starch solution, 1% (m/V)
4. apparatus and EQUIPMENT
4.1 the beaker on the 100 ml
4.2 50 ml Burette
4.3. volumetric flasks, 250 ml
4.4. Measuring cylinders, 25 and 100 ml
4.5 Not pipette 10 ml
4.6. conical flasks, 250 ml
5. the procedure
5.1
Into a 100 ml beaker weigh about 10 g (m gram) of sample exactly
containing about 0.6 g of hydrogen peroxide. The content is using the water converts into
the 250 ml volumetric flask, make up to the mark with water and mix.
5.2.
10 ml of the sample solution (5.1), into a conical flask transfer by pipette on 250
ml (4.6) and add successively 100 ml 4 mol/l sulphuric acid (3.1), 20
ml of potassium iodide solution (3.5) and three drops of a solution of molybdate
(3.6).
5.3.
Titrate the iodine formed immediately 0.2 mol/l sodium Thiosulphate solution
(3.4) and just before reaching the equivalence point, add a few
millilitres of starch solution as indicator (3.7). Record the consumption of
0.2 mol/l sodium Thiosulphate (3.4) in millilitres (V).
5.4.
In the manner described in sections 5.2 and 5.3, carry out a blank test taking
replacing the 10 ml of the sample solution 10 ml of water. Record the consumption of 0, 1N
solution of Sodium Thiosulphate in the blank (V0 ml).
6. The CALCULATION of the
The content of hydrogen peroxide in the product, expressed as percentage by weight (%
m/m) is calculated using the following formula:
(V-V0) x 1.7008 x 250 x 100
% hydrogen peroxide =--------------------------------
m x 10 x 1000
(V-V0) x 4.252
= --------------------
m
in which:
m = amount of test sample (5.1) in grams,
V0 = consumption 0.2 mol/l sodium Thiosulphate in the blank
attempt (3.4) in millilitres,
In = consumption 0.2 mol/l sodium Thiosulphate solution in the
titration of the sample solution (5.3) in millilitres.
7. REPEATABILITY
For products containing about 6% (m/m) of hydrogen peroxide the difference between the
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.2%.
9. qualitative and SEMI-QUANTITATIVE DETERMINATION OF CERTAIN OXIDATION
COLORANTS IN HAIR DYES
1. scope and field of application
This method is suitable for the qualitative and semi-quantitative determination of
the following substances in hair dyes in cream or liquid form:
------------------------------------------------
The Substance Symbol
------------------------------------------------
Phenylenediamines
ofenylendiamin (OPD)
mfenylendiamin (MPD)
pfenylendiamin (PPD)
------------------------------------------------
Methylphenylenediamines
4methyl1, 2fenylendiamin (OTD)
(toluen3, 4diamin)
4methyl1,-m-Phenylenediamine (MTD)
(toluen2, 4diamin)
2-methyl, 4fenylendiamin (PTD)
(toluen2, 5diamin)
------------------------------------------------
Diaminofenoly
2, 4diaminofenol (DAP)
Hydroquinone
Benzen1, 4diol (H)
alfanaftol (alfaN)
------------------------------------------------
Pyrogallol
Benzene-1, 2,3-triol (P)
------------------------------------------------
Resorcinol
Benzene-1.3-diol (R)
------------------------------------------------
2. The PRINCIPLE of the
Oxidation dyes are extracted from the colors in the form of a cream or liquid when
Ph 10 96% ethanol, and identify one or two-dimensional
thin-layer chromatography.
The semikvantitativnímu determination of these substances is a chromatogram of samples
compares using the four developing systems with reference chromatograms
substances taken by at the same time and under what conditions nejpodobnějších.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Ethanol, anhydrous
3.2 Acetone
3.3. Ethanol, 96% (V/V)
3.4. Ammonia solution, 25% (d204 = 0.91)
3.5 L (+)-Ascorbic acid
3.6 Chloroform
3.7 Cyclohexane
2.4 nitrogen, technical grade
2.4 Toluene
3.10 Benzene
3.11 Butan1ol
3.12 Butan2ol
3.13 fosforná Acid, 50% solution (in/in)
3.14 Diazotační reagent.
Either:
. 3nitro1benzendiazoniumchlorbenzensulfonát (in the form of stable salts)
as for example. in red 2 JN-Francolor,
. 2chlor4nitro1benzendiazoniumnaftalenbenzoát (in the form of stable salts)
as for example. in NNCD reagent-reference no 74 150 FLUKA,.
or equivalent.
3.15. silver nitrate
3.16 pdimethylaminobenzaldehyd
3.17 2, 5dimethylfenol
Ferric Chloride, hexahydrate 3.18
3.19. hydrochloric acid, 10% solution (m/V)
3.20 Standards
Standards are the compounds referred to in section 1 "purpose and scope".
In the case of aminosloučenin must be either in the form of a standard compound
hydrochloride (mono or di), or as the free base.
3.21 standard solutions 0.5% (m/V)
Prepare a 0.5% solution (m/V) the standards referred to in paragraph 3.20.
Weigh out 50 mg +/-1 standard to 10 ml volumetric flasks.
Add 5 ml of 96% ethanol (3.3) and 250 ml of the Ascorbic acid solution (3.5).
The solution is alkaline by the addition of the ammonia solution (3.4) on the value of pH 10
(retrieves the indicator paper).
Make up to 10 ml of 96% ethanol (3.3) and mix.
Solutions can be kept for a week in a cool and dark place.
In some cases, after the addition of Ascorbic acid and the solution
ammonia arise. Before proceeding you must let her
settle down.
3.22. mobile phase
acetone-chloroform-toluene (35:25:40, V/V/V)
3.22.2 Chloroform-cyclohexane-ethanol-25% ammonia solution (80:10
: 10:1, V/V/V/V)
3.22.3 Benzene-butan2ol-water (50:25:25, V/V/V).
Shake vigorously and after separating at room temperature (20 to 25th.
(C)) to use the upper phase.
3.22.4. nbutanol-chloroform-reagent M (7:70:23, in/in/in).
Carefully dekantují phase at room temperature (20 to 25 ° C) and
the lower phase is used.
Preparation of the reagent M
Ammonia solution, 25% (V/V) 24 volumes of
Fosforná acid, 50% (3.13) 1 bulk unit
Water vol. 75 units
Note:
The mobile phase containing ammonia must be immediately before use
shake well.
3.23 the detection reagents
3.23.1 Diazotační reagent
Prepare a 5% (m/V) aqueous solution of the chosen reagent (3.14). This
solution must be prepared immediately before use.
3.23.2 Ehrlichovo reagent
2 g pdimethylaminobenzaldehydu (3.16) is dissolved in 100 ml of 10% aqueous
hydrochloric acid solution (m/V) (3.19).
3.23.3 2, 5dimethylfenol-ferric chloride hexahydrate
Solution 1: dissolve 1 g of dimethylfenolu (3.17) in 100 ml of 96% ethanol
(3.3).
Solution 2: dissolve 4 g of ferric chloride hexahydrátu (3.18) in 100
ml of 96% ethanol (3.3).
When invoking the chromatogram with these solutions done by spraying
separately, first solution 1, then the solution 2.
3.23.4 Ammoniacal Silver nitrate
25% ammonia (3.4) is added to a 5% (m/V) aqueous solution of nitrate
Silver (3.15) until the precipitate just dissolves. This reagent
is prepared immediately before use. It cannot be kept.
4. apparatus and EQUIPMENT
4.1. Usual laboratory equipment for thin-layer chromatography.
4.1.1
Plastic or glass cover designed to chromatography
the plate during the application of stains and drying could wrap the nitrogen. This measure
It is necessary due to the tendency of certain dyes to oxidation.
4.1.2
Microsyringe, 10 mikrol, graduated in 0.2, mikrol with a needle with equal
at the end, or a better dispenser 50 mikrol clamped in the holder so that the
the plate can be kept under nitrogen.
4.1.3.
Ready made silica gel plates, 0.25 mm thickness, dimensions 20 x 20 cm
(Macherey and Nagel, Silica GHR, the plastic backing, or
equivalent).
4.2. centrifuge, 4000 RPM.
4.3 10 ml centrifuge tube with screw fasteners coated with
PTFE or equivalent.
5. the procedure
5.1. Preparation of the sample for analysis
The first 2 to 3 cm of cream beaten from the tube is removed.
Into the centrifuge tube (4.3) previously flushed out with nitrogen is converted:
300 mg Ascorbic acid with 3 g cream or 3 g homogenized
the liquid.
Adds drop 25% ammonia (3.4) until the pH reaches the value of 10.
Make up to 10 ml of 96% ethanol (3.3).
Homogenize under nitrogen (3.8), stopper and
is centrifuged after 10 minutes at 4000 RPM.
The supernatant liquid shall apply.
5.2 Chromatography
5.2.1 Coating on plate
Under the atmosphere of nitrogen (3.8) on a chromatography plate (4.1.3)
apply after 1 mikrol each of the above reference substance in the nine points
1.5 cm apart along the line about 1.5 cm from the edge
the boards.
The reference substances are used in this arrangement:
1 2 3 4 5 6 7 8 9
R P H PPD DAP PTD OPD OTD MPD
MTD alfaN
In addition, in paragraphs 10 and 11 of the TLC always 2 mikrol investigated solutions
obtained according to section 5.1.
Keep the plate under nitrogen (3.8) until the moment when it is
chromatographed.
5.2.2 Develop
The Board is inserted into the Chamber previously flushed out with nitrogen (3.8), saturated
one of the four solvents (3.22) and allow it to develop in the laboratory
temperature (20 to 25 ° C) in the dark until the solvent front reaches the
a distance of about 15 cm from the starting line.
The plate and dried under nitrogen (3.8) in the laboratory
temperature.
5.2.3 the invocation
Spray the plate with the corresponding detection agent referred to in point
3.23.
5.2.4 the Quality determination
Compare the Rf values and colours stain sample and interpret the
reference substances.
Table 1 lists examples of Rf values and colours of the spots for
each substance depending on the solvent and the indicator.
Uncertain evidence is sometimes can be confirmed by the method of standard product.
5.2.5 Semi-quantitative estimation
The intensity of the spots of each of the substances identified in item 5.2.4 is
compares the Visual with the corresponding range of concentrations of the standard
substances.
When the excessively high concentration of one or more of the substances found in the
the sample is a sample extract diluted and the measurement repeated.
Table 1
The Rf values and colours obtained immediately after invoking the
----------------------------------------------------------------------------------------------
| Standard | The mobile phase | The mobile phase |
| (3.20) | | |
| | The Rf Values | The resulting colors |
| | (3.22.1) | (3.22.2) | (3.22.3) | (3.22.4.) | Diazo | Ehrlich | Dimethylphenol | Agno3 |
| | | | | | | | (3.23.3) | |
| | | | | | (3.23.1) | (3.23.2) | | (3.23.4) |
-----------------------------------------------------------------------------------------------
| OPD | 0.62 | 0.60 | 0.30 | 0.57 | light |-|-| light brown |
| | | | | |-Brown | || |
| MPD | 0.40 | 0.60 | 0.47 | 0.48 | violet | yellow | | light brown light brown |
| | | | | |-Brown (*) | || |
| PPD | 0.50 0.30 0.20 | | | | | 0.48 Brown purple grey clear | | |
| | | | | | |-Red | |
| | | | | | | (*) || |
| 0.60 0.60 OTD | | | 0.53 | 0.60 | Brown (*) light | Auburn | greyish Brown |
| | | | | | | Orange | |
| MTD | 0.40 | 0.67 | 0.45 | 0.60 | reddish | yellow | brown | black |
| | | | | |-Brown (*) | || |
| PTD | 0.33 | 0.65 0.70 0.37 | | | | | orange brown purple grey * | |
| DAP | |-0.07 | 0 | 0.05 | Brown (*) | Orange | Purple | Brown |
| H | 0.50 | 0.35 | 0.80 | 0.20 |-| | |-black purple orange (*) |
| | | 0.80 0.90 alfaN | 0.90 | 0.75 | Orange |-|-violet (*) black |
| | | | | |-Brown | || |
| P | |-0.37 | 0.67 | 0.05 | Brown very very light ' | | |-Brown (*) |
| | | | | | light brown | | | |
| | | | | | |-Violet | |
| R | 0.50 | 0.37 | 0.80 | 0.17 | orange | light | | very light light brown |
| | | | | | (*) purple | brown | |
----------------------------------------------------------------------------------------------
Note 1. OPD is showing only slightly; the solvent must be used (3.22.3), in order to clearly distinguish it from OTD. |
2. (*) indicates best induced by color.
6. TEST of TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY
This method of two-dimensional chromatography requires the use of additional
standards and reagents.
6.1 Additional standards and reagents
6.1.1 betanaftol (betaN)
6.1.2 2aminofenol (OAP)
6.1.3.3aminofenol (MAP)
6.1.4 4aminofenol (PAP)
6.1.5 2nitro1, 4fenylendiamin (2NPPD)
6.1.6 4nitro1, 2fenylendiamin (4NOPD)
Prepare a 0.5% (m/V) solutions of each of the other standards in the manner
described in paragraph 3.21.
6.2 other mobile phase
Ethyl acetate-cyclohexane-6.2.1 25% ammonia solution (65:30:0.5,
V/V/V)
6.3 Additional detection system
In a tank for thin-layer chromatography with glass inserts
the container, add about 2 g crystallized iodine and the Chamber is closed
the matching lid.
6.4 Chromatography
6.4.1
As shown in Figure 6 is on the plate for thin-layer chromatography (4.1.3)
be marked on the side with two adsorbentem lines.
6.4.2
In a nitrogen atmosphere (4.1.1), apply 1 to 4 mikrol extract (5.1) into the
the base point 1 (Figure 6), which is about 2 cm from the edge.
The amount of extract depends on the intensity of the spots on the chromatograms by
5.2.
6.4.3
Between points 2 and 3 (fig. 6) the oxidation colorants identified plated
or considered to be identified by 5.2 (distance between points
is 1.5 cm). Applied with 2 mikrol of each reference solution-in addition to
The DAP, which is to be applied in volume 6 of the mikrol. The application shall be carried out in
the atmosphere nitrogen (6.4.2).
6.4.4
The procedure referred to in section 6.4.3 shall be repeated in the base points 4 and 5 (fig. 6) and
keep the plate under nitrogen until raising the chromatogram
(distance between points 1.5 cm).
6.4.5
Chromatography Chamber is purged with nitrogen (3.8) and place in it
appropriate amount of mobile phase 3.22.2. Plate (6.4.4) is inserted into the Chamber and
develops in the dark in the first development direction (fig. 6).
Develop until the solvent front reaches the line marked on the motherboard
(approximately 13 cm).
6.4.6
The plate from the tank and placed into a different chromatographic Chambers
previously flushed out with nitrogen, to developing solvent odpařovalo
at least 60 minutes.
6.4.7
Using the graduated test tube is flushed out in advance into the Chamber with nitrogen (3.8)
bring the appropriate amount of the mobile phase (3.9), into the Chamber insert plate
rotated 90 St. (6.4.6) and chromatogram in the other direction (again,
in the dark) until the solvent front reaches the line marked on the side with the
adsorbentem. The plate from the tank and allow the solvent to evaporate on the
the air.
6.4.8
The plate is placed at 10 minutes to the chromatography tank containing steam
iodine (6.3) and chromatogram is interpreted using the
Rf values and colours simultaneously interpret the standards (the Rf values and
the colors of the spots are listed in table II).
Note
The best paint stain is achieved if the chromatogram exposed after developing
chromatogram in the atmosphere a vapour of iodine for half an hour.
6.4.9
The presence of the oxidation colorants found in accordance with section 6.4.8 can be
definitively confirmed by repeating the procedure described in paragraphs 6.4.1 to
6.4.8 when in the base point 1 through the amount of extract in accordance with point
6.4.2 spotted 1 mikrol of the reference substances identified in 6.4.8 point.
Neobjevíli is compared with the chromatogram obtained as described in section 6.4.8 no
Another bright spot is the interpretation of chromatogram 6.4.8 is correct according to the point.
Table II
The colors of the spots after chromatography standards and developing iodine vapours
------------------------------------------------
Colors standards after developing iodine vapours
------------------------------------------------
R beige
P Brown
Alpha N purple
betaN light brown
(H) violet/Brown
MPD yellowish brown
PPD violet Brown
MTD dark brown
PTD yellowish brown
DAP dark brown
OAP Orange
MAPS of yellowish brown
PAP violet Brown
2NPPD Brown
4NOPD Orange
------------------------------------------------
10. The qualitative and QUANTITATIVE DETERMINATION of NITRITE
AND QUALITATIVE DETERMINATION
1. scope and field of application
This method is suitable for qualitative determination of nitrite in
cosmetic products, in particular in creams and toothpastes.
2. The PRINCIPLE of the
The presence of nitrite indicates the formation of coloured derivatives with 2-
aminobenzaldehydfenylhydrazonem (Nitrin (R)).
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 the dilute sulphuric acid:
2 ml of the concentrated sulphuric acid (d204 = 1.84) with 11 ml
distilled water.
3.2. dilute hydrochloric acid:
1 ml of concentrated hydrochloric acid (d204 = 1.19) diluted 11 ml
distilled water.
3.3 Methanol
2aminobenzaldehydfenylhydrazonu solution (reagent Nitrin (R))
in methanol
Take 2.0 g Nitrinu (R), transfer quantitatively into a graduated flask
the 100 ml. Add a drop of 4 ml of dilute hydrochloric acid
(3.2) and mix. Make up to the mark with methanol and mix until
the solution completely. Keep the solution in a brown glass bottle (4.3).
4. apparatus and EQUIPMENT
4.1 in a 50 ml Beaker
4.2 the volumetric flask 100 ml
4.3 Brown on 125 ml glass bottle
4.4 the glass plate, 10 x 10 cm
4.5 the Spatula, plastic
4.6 filter paper, 10 x 10 cm
5. the procedure
5.1
Part of the sample evenly over the glass plate
(4.4) so that the surface was covered with a layer of no more than 1 cm thick.
5.2.
Sheet of filter paper (4.6) soak with distilled water. Lay it
on the pattern and attach a plastic spatula (4.5).
5.3.
Wait for about 1 minute, and in the center of the filter paper with TLC:
. 2 drops of dilute sulphuric acid (3.1),
. then two drops of Nitrinu (R) (3.4).
5.4.
After 5 to 10 seconds, with the filter paper removed and inspected against the
daylight. A reddish purple coloration indicates the presence of nitrite.
Contents of nitrite drove a low, red-violet coloring changes during 5 to
15 seconds on the yellow. They were driving a large amount of nitrite are present, this occurs
color change after 1 or 2 minutes.
6. the NOTE
The intensity of the violet colour and the time that elapses before the change to the
yellow, suggest the content of nitrite in the sample.
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
The method describes the quantitative determination of nitrite in cosmetic
resource.
2. The DEFINITION of the
Nitrite content of the sample, as determined by this method is expressed in
percentage by weight of sodium nitrite.
3. The PRINCIPLE of the
After diluting the sample with water and allow to be clarification responses
nitrite is present, sulfanilamidem, and N1naftylethylendiaminem and
the absorbance of the resulting coloring is measured at 538 nm.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1 Čeřidla:
These agents cannot be used more than one week after preparation.
4.1.1. carrez solution I:
106 (g) hexacyanoferrate (ii) K4Fe (CN) 63H2O in
distilled water and dilute with water to 1000 ml.
4.1.2. carrez solution II:
219.5 g of zinc acetate Zn (CH3COO) 2.2H2O and 30 ml of glacial acid
acetic acid is dissolved in distilled water and dilute with water to 1000 ml.
sodium nitrite solution:
0.500 g of sodium nitrite in distilled water in a volumetric flask
on the 1000 ml and make up to the mark with water. 10.0 ml of the stock
dilute the standard solution to 500 ml; 1 ml of the resulting solution = 10
mikrog NaNO2.
4.3. sodium hydroxide solution, 1 mol/l
4.4. The sulfa-hydrochloride Solution, 0,2%:
2.0 g sulfanilamidu for heating to dissolve in 800 ml of water. After
cooling with stirring add 100 ml concentrated acid
hydrochloric. Dilute with water to 1000 ml.
2.8. hydrochloric acid, 5 mol/l
4.6 N1naftylové reagent:
This solution must be prepared on the day of use. Dissolve 0.1 g
Nnaftylethylendiaminu dihydrochloride in water and dilute with water to 100
ml.
5. apparatus and EQUIPMENT
5.1. analytical balance
5.2. Graduated flasks of 100, 250, 500 and 1000 ml
5.3. bulb or graduated pipettes
5.4. Measuring cylinders, 100 ml
5.5 fluted filter paper, no nitrite, diameter 15 cm
5.6 the waterbath
5.7 the Spectrophotometer and the cells with 1 cm optical path
5.8 pH meter
5.9 the Microburette of 10 ml
5.10 250 ml Beaker to
6. the procedure
6.1
To the nearest 0.1 mg approximately 0.5 g (m gram) of the homogenized
sample, transfer quantitatively to a beaker with distilled water, hot
the 250 ml (5.10) and make up to volume with distilled water on hot about 150 ml.
Beaker (5.10) for half an hour into the waterbath (5.6) heated to
80. C. During this time, shake the contents occasionally.
6.2.
Cool to room temperature and gradually with stirring add 2 ml
Carrez I reagent (4.1.1) and 2 ml of carrez solution II (4.1.2).
6.3.
Adds 1 mol/l sodium hydroxide solution (4.3) until the pH is not adjusted
at 8.3. (For use with pH meter (5.8)). Transfer quantitatively into a
the 250 ml volumetric flask (5.2) and make up to volume with distilled water.
6.4.
Mix and filter through a fluted filter paper (5.5).
6.5.
In a 100 ml volumetric flask (5.2), transfer by pipette (5.3) of the corresponding
an aliquot portion (in millilitres) of the clear filtrate, but not more than 25 ml,
and make up to volume with distilled water to a volume of 60 ml.
4.1
After mixing, add 10.0 ml of sulfa-hydrochloride (4.4)
and then 6.0 ml of 5 mol/l hydrochloric acid (4.5). Mix and
stand for 5 minutes. Add 2.0 ml N1naftylového reagent (4.6),
mix and leave to stand for 3 minutes. Dilute with water to the mark and
mix.
4.2
Prepare a blank by repeating the operations described in points 6.5 and 6.6
without the addition of N1naftylového reagent (4.6).
4.2
Measure the absorbance of the solution (5.7), prepared in accordance with section 6.6 in 538
nm from the blank solution (4.2).
4.3
From the calibration graph (6.10) minus the content of sodium nitrite in
micrograms per 100 ml of the solution (m1 micrograms), corresponding to the absorbence
measured according to paragraph 4.2.
6.10
Using a solution of sodium nitrite concentration 10 mikrog/ml (4.2)
plot a calibration graph for concentrations of 0, 20, 40, 60, 80 and 100 mikrog
sodium nitrite in 100 ml.
7. The CALCULATION of the
Sodium nitrite content of the sample, expressed as percentage by weight is
calculated using the following formula:
250 100 m1
% NaNO2 =----x m1 x 10-6 x--------------
In the m x m x 40
where:
m = mass of the sample taken for analysis in g (6.1),
M1 = the content of sodium nitrite in mikrog established pursuant to section 4.3,
In = the number of millilitres of filtrate is used for measurement (6.5).
8. REPEATABILITY
For products containing about 0.2% (m/m) of sodium nitrite the difference between the
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.005%.
11. The QUANTITATIVE DETERMINATION of RESORCINOL in shampoos and hair
LOTIONS
1. scope and field of application
In this method, the quantitative determination of resorcinol is specified in
shampoos and hair waters by gas chromatography. The method is suitable
for the concentration of 0.1 up to 2.0% by weight of the sample.
2. The DEFINITION of the
The content of resorcinol in the sample, as determined by this method is expressed in
percentage by weight.
3. The PRINCIPLE of the
Resorcinol and 3,5-dihydroxytoluene, 3, (5methylresorcinol), to be added as the
internal standard, from the sample separated by thin layer chromatography.
Both compounds are isolated by scraping their spots from the upper layer of the plate
and extraction with methanol. Finally the extracted compounds are dry,
silylated and determined by gas chromatography.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Hydrochloric acid solution, 25% (m/m)
4.2. Methanol
4.3. Ethanol, 96% (V/V)
4.4.
Ready made silica gel TLC plates (plastic or aluminium) with
fluorescence indicator. The Board shall recognize the following:
silikagelová the plate is sprayed with water until glazed.
The sprayed plates to dry at room temperature for one to
three hours.
Note
Without the recognition of losses of resorcinol can occur as a result of
irreversible adsorption on silica gel.
4.5. Developing solvent:
acetone-chloroform-acetic acid (20:75:5; V/V/V).
4.6. resorcinol standard solution:
dissolve 400 mg resorcinol in 100 ml of 96% ethanol (4.3) (1 ml
corresponds to 4000 mikrog resorcinol).
4.7. Internal standard solution:
dissolve 400 mg 3, 5dihydroxytoluenu (DHT) in 100 ml of 96% ethanol
(4.3) (1 ml corresponds to 4000 mikrog DHT).
4.8 a mixture of standards:
in a 100 ml volumetric flask, mix 10 ml of solution 4.6 and 10 ml of solution
4.7, make up to the mark with 96% ethanol (4.3) and mix (1 ml of the solution
corresponds to 400 mikrog 400 mikrog resorcinol and DHT).
4.9 Silylating agents:
4.9.1 N, Obis (trimethylsilyl) trifluoracetamid (BSTFA)
4.9.2 Hexamethyldisilazan (HMDS)
4.9.3 Chlortrimethylsilan (TMCS)
5. apparatus and EQUIPMENT
5.1 Normal equipment for thin-layer chromatography and gas
chromatography
5.2 Glass laboratory dishes
6. the procedure
6.1. Preparation of the sample
6.1.1.
150 ml beaker weigh accurately a test sample (m grams)
the device, which contains about 20 to 50 mg resorcinol.
6.1.2.
Acidify with hydrochloric acid (4.1) into a sour response (you need
approximately 2 to 4 ml), add 10 ml (40 mg DHT) of the internal
standard (4.7) and mix. With ethanol (4.3), volumetric
100 ml flask, make up to the mark with ethanol and mix.
6.1.3
250 mikrol of the solution (6.1.2) to a deactivated plate
silica gel (4.4) as a continuous line of approximately 8 cm length.
care should be taken to that line was the closest.
6.1.4.
On the same plate in the same way (6.1.3) spotted 250 mikrol mixture
standards (4.8).
6.1.5
Within two points of the starting line 5 mikrol deposit of the solutions 4.6 and 4.7 to
facilitate the location of the spots after the invocation.
6.1.6
The Board develops in the unsaturated Chamber filled with developing solvent
(4.5) until the solvent front has reached a distance of 12 cm from the
the starting line; usually, it takes about 45 minutes. The plate is dried on
the air and the zone of resorcinol/DHT under short wave UV
light (254 nm). Both of these compounds have approximately the same value
RF bands. shall be marked with a pencil at a distance of 2 mm from the outer
Dark interface. These zones are removed from the Board and the adsorbent
each belt is collected in a bottle of 10 ml.
6.1.7
Adsorbent containing the sample and the adsorbent containing a mixture of standards,
extract the following:
Add 2 ml methanol (4.2) and extract for one hour
continuous mixing. Filter the mixture and repeat the extraction step for
the next 15 minutes with 2 ml methanol.
6.1.8.
Extracts combine and allow the solvent to evaporate overnight in a vacuum
desiccator of Silicagel. Nezahřívat.
6.1.9 (6.1.8) silylate, as described in paragraph 6.1.9.1 or
6.1.9.2.
6.1.9.1 Microsyringe, add 200 BSTFA (4.9.1) mikrol and the mixture is
leave in a closed vessel for 12 hours at room temperature.
6.1.9.2 Microsyringe gradually adds 200 HMDS (4.9.2) mikrol and 100
mikrol TMCS (4.9.3), the mixture is heated in a closed container for 30 minutes at 60
St. c. mixture is cooled.
6.2 gas chromatography
6.2.1. Chromatographic conditions
The column must provide resolution R equal to, or better than, 1.5, where:
2 d ' (r2-r1)
R = -----------------
W1 + w2
where:
R1 and r2 = retention times, in minutes, of two of the peaks,
W1 and w2 = peak widths at half height, in millimetres,
d ' = the chart speed in mm/min.
The following column and conditions for gas chromatography have shown
as appropriate:
Column material: stainless steel
Length 200 cm
Internal diameter: approx. 3 mm
Filling: OV17 10% on chromosorb WAW 100 to 120 mesh
Flame ionization detector
Temperature mode:
Column: 185 ° C (determination)
Detector: 250 ° C
Spray: 250 ° C
Carrier gas: nitrogen
Flow rate: 45 ml/min
Hydrogen and air flow rates are set according to the manufacturer's instructions.
6.2.2.
Into the gas chromatograph inject 1 to 3 mikrol solutions
prepared in accordance with section 6.1.9. For each solution (6.1.9) shall be five
injections, measure the peak areas, calculate the average peak area ratio:
= Peak area resorcinol/peak area DHT.
7. The CALCULATION of the
The concentration of resorcinol in the sample, expressed as percentage by weight
(% m/m), is given by:
4 Svzorku
where:% =--resorcinol-x--------------------
M Sstandardní mixture
M = mass of test sample in g (6.1.1)
Svzorku = the average peak area (6.2.2) for solution
of the sample,
Sstandardní of the mixture = the average peak area (6.2.2) for a mixture of
standards.
8. REPEATABILITY
For products containing about 0.5% (m/m) the difference between the resorcinol
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.025%.
12. The QUANTITATIVE DETERMINATION of METHANOL in RELATION to ethanol or PROPAN-
2OLU
1. scope and field of application
The method is described in the analysis of methanol in all types of cosmetic
resources (including aerosols) by gas chromatography.
Method is to determine the relative levels of 0 to 10%.
2. The DEFINITION of the
The content of methanol, as determined by this method is expressed in
percentage of methanol in relation to ethanol or propan2olu.
3. The PRINCIPLE of the
The determination is done by gas chromatography.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1 Methanol
4.2. ethanol, Absolute
4.3 Propan2ol
Chloroform, freed from alcohols by washing 4.4 water
5. apparatus and EQUIPMENT
5.1 gas chromatograph:
with katharometer detector (katarometr) for aerosol samples,
with flame ionization detector for non-aerosol samples.
5.2. volumetric flasks, 100 ml
5.3. Pipettes of 2 ml, 20 ml and 0 to 1 ml
5.4 the Microsyringe for 0 to 100 mikrol and 0 to 5 mikrol
(only for aerosol samples) special gastight syringe
with sliding valve (see Figure 5, chapter "preparation of the sample in the
the lab ").
6. the procedure
6.1. Preparation of the sample
6.1.1.
Samples of the resources in the package to aerosols shall be taken in accordance with the procedure
the chapter "preparation of samples in the laboratory and then analysed by gas
chromatography under the conditions described in point 6.2.1.
6.1.2.
Samples other than aerosol resources subscribed in accordance with the procedure
the above chapter "preparation of samples in the laboratory", with water on the
a concentration of 1 to 2% ethanol or propan2olu, and then analyze the
gas chromatography under the conditions described in 6.2.2.
6.2 gas chromatography
6.2.1 For aerosol samples uses the thermal conductivity detector
(katarometr).
6.2.1.1 Filling of the column is 10% Hallcomid M18 on chromosorb WAW 100 to
200 mesh.
6.2.1.2. The column must provide resolution R equal to, or better than, 1.5,
where:
d ' r2-d ' r1
R = 2 -------------
W1 + w2
where:
R1 and r2 = retention times, in minutes, of two of the peaks,
W1 and w2 = peak widths at half height, in millimetres,
d ' = the chart speed in mm/min.
6.2.1.3 tab of this resolution can be achieved under the following conditions:
Column material: stainless steel
Length: 3.5 meter
Diameter: 3 mm
The current bridge katarometru: 150 mA
Carrier gas: helium
Pressure: 2.5 bar
Flow rate: 45 ml/min
Temperature mode:
Injector: 150 ° C
Detector: 150 ° C
Column: 65 ° C
Measure the peak areas, it is possible to improve the electronic integration.
6.2.2 other than aerosol samples:
6.2.2.1 the Filling of the column is Chromosorb 105 or Porapak QS; It shall apply
the flame ionization detector.
6.2.2.2. The column must be a resolution equal to or better than the R 1.5, where:
d ' r2-d ' r1
R = 2 -------------
W1 + w2
R1 and r2 = retention times, in minutes, of two peaks.
W1 and w2 = peak widths at half height, in millimetres,
d ' = the chart speed record in mm/min.
6.2.2.3 This resolution can be achieved under the following conditions:
Column material: stainless steel
Length: 2 meters
Diameter: 3 mm
The sensitivity of the elektrometru: 8x1010 and
Gases:
Carrier gas: nitrogen
Pressure: 2.1 bar
Flow: 40 ml/min
Auxiliary gas: hydrogen
Pressure: 1.5 bar
Flow: 20 ml/min
Temperature mode:
Injector: 150 ° C
Detector: 230 ° C
Column: 120 to 130 ° C
7. CALIBRATION GRAPH
7.1
For gas chromatography procedure 6.2.1 (Hallcomid
M18) use the following standard mixtures. These mixtures are prepared
metering by using pipettes, but the exact amount will be determined by weighing the pipettes
or flask after each addition.
-------------------------------------------------------
Ethanol or Methanol followed by relative
concentration (ml) of chloroform on propan2ol
(% m/m): volume (ml)
-------------------------------------------------------
Approximately 20 100 0.5 (ml)
2.5%
-------------------------------------------------------
Approximately 1.0 20 100 (ml)
5.0%
-------------------------------------------------------
Approximately 1.5 20 100 (ml)
7.5%
-------------------------------------------------------
Approximately 2.0 20 100 (ml)
10.0%
-------------------------------------------------------
Into the chromatograph is nastřikují 2 to 3 mikrol under the conditions referred to in
6.2.1.
The mixture shall be calculated for each of the ratios of the peak areas (methanol/ethanol) or
(methanol/propan2ol). Plot a calibration graph plotting
on the x axis: the amount of methanol in relation to ethanol or propan2olu in%
on the y-axis: peak area ratio (methanol/ethanol) or (methanol/propan-
2ol).
7.2.
For gas chromatography procedure 6.2.2 (Porapak QS
or Chromosorb 105) use the following standard mixtures. These
the mixture is prepared by using the metering of the pipettes, but the exact amount of the
provides for weighing of the pipette or flask after each addition.
----------------------------------------------------
Ethanol or Methanol followed by relative
concentration (mikrol) propan2ol water on the
(% m/m): volume (ml)
----------------------------------------------------
Approximately 50 2 100 (ml)
2.5%
----------------------------------------------------
Approximately 100 2 100 (ml)
5.0%
----------------------------------------------------
Approximately 150 2 100 (ml)
7.5%
----------------------------------------------------
Approximately 200 2 100 (ml)
10.0%
----------------------------------------------------
Into the chromatograph is nastřikují 2 to 3 mikrol under the conditions referred to in
6.2.2.
The mixture shall be calculated for each of the ratios of the peak areas (methanol/ethanol) or
(methanol/propan2ol), and the calibration graph plotting
on the x axis: the amount of methanol in relation to ethanol or propan2olu in%
on the y-axis: peak area ratio (methanol/ethanol) or (methanol/propan-
2ol).
7.3 calibration graph should be linear.
8. REPEATABILITY
For products containing relatively about 5% (m/m) of methanol, given the
ethanol or propan2olu the difference between the results of two parallel
determination on the same sample should not exceed an absolute value of 0.25%.
13. The qualitative and quantitative DETERMINATION of FREE FORMALDEHYDE
1. scope and field of application
This method describes the determination of quality and two ways
quantitative determination of depending on whether or not they are
formaldehyde donors present. Is applicable to all cosmetic
resources and consists of three parts:
1.1. Qualitative determination
1.2 General determination by pentane, 4dionu
(acetylaceton)
This method is used in case when used alone or combined
with preservatives that are not formaldehyde donors.
Otherwise, and if the result exceeds the maximum permitted
the concentration, the following method must be used for confirmation.
1.3 quantitative determination in the presence of formaldehyde donors
When using the above methods (1.2) when derivatisation donors
formaldehyde break down, which leads to too high results (bound
formaldehyde and formaldehyde in polymer form). First, free
formaldehyde (or bound in a polymeric form) liquid chromatography.
2. The DEFINITION of the
Free formaldehyde content of the sample determined by this method is expressed as
the percentage by weight.
3. QUALITATIVE DETERMINATION
3.1 the principle
Free and bound formaldehyde in a sulphuric acid
the presence of Schiff's reagent pink or mauve coloring.
3.2 USED CHEMICALS
All the reagents must be of analytical purity or higher and uses the
with demineralized water.
3.2.1 Fuchsin
3.2.2. Sodium Sulphite heptahydrate
3.2.3 concentrated hydrochloric acid
(d204 = 1.19)
3.2.4. Sulphuric acid solution, approximately 1 mol/l
3.2.5 the Schiff's reagent:
Beaker, weigh 100 mg of fuchsin (3.2.1) and dissolved in 75 ml of water
at a temperature of 80 ° C. After cooling, add 2.5 g of sodium sulphite
heptahydrátu (3.2.2) and 1.5 ml of hydrochloric acid (3.2.3). The volume of the
make up to 100 ml.
Application within two weeks.
3.3 Procedure
3.3.1
In a 10-ml beaker weigh out 2 g of the sample.
3.3.2.
Add 2 drops of sulphuric acid (3.2.4) and 2 ml of Schiff's reagent
(3.2.5). This reagent must be absolutely colourless when used.
The contents of the shake and leave to stand for 5 minutes.
3.2.3.
The hatchway into the 5-minute pink coloring, or the color mauve, is present
formaldehyde in amounts greater than 0.01% and must be set free, and
bound formaldehyde in accordance with point 4. and, if necessary, in accordance with section 5.
4. the GENERAL DETERMINATION of FORMALDEHYDE with Pentane, 4DIONU
4.1 Principle formaldehyde reacts with pentane, in the presence of acetate
for making 3, 5diacetyl1, 4dihydrolutidinu. The extracted
butan1olem and the absorbance of the extract is measured at 410 nm.
4.2 USED CHEMICALS
All the reagents must be of analytical purity or higher and uses the
with demineralized water.
4.2.1 Anhydrous ammonium acetate
concentrated acetic acid,
(d204 = 1.05)
4.2.3 Pentane-freshly distilled under reduced pressure 25 mm Hg
at 25 ° C – not exhibit any absorption at 410 nm
4.2.4 Butane-1-ol
4.2.5. hydrochloric acid, 1 mol/l
4.2.6. hydrochloric acid, approximately 0.1 mol/l
4.2.7. Sodium hydroxide, 1 mol/l
4.2.8, freshly prepared starch solution (1 g/50 ml of water) by the European
Pharmacopoeia, 2. Edition 1980, part of the IVI11
4.2.9 37% to 40% (m/V) solution of formaldehyde
4.2.10 Standard iodine solution, 0.05 mol/l
4.2.11 standard Sodium Thiosulphate solution, 0.1 mol/l
4.2.12 Reagent containing pentane-:
In a 1000 ml volumetric flask dissolve:
-150 g ammonium acetate (4.2.1),
-2 ml pentane, 4dionu (4.2.3),
-3 ml acetic acid (4.2.2).
Make up to 1000 ml with water (pH of solution about 6,4).
This reagent must be freshly prepared.
4.2.13 Reagent (4.2.12) without the addition of pentane, 4dionu
4.2.14 the formaldehyde standard solution: stock solution
5 g of formaldehyde (4.2.9) into a volumetric flask of 1000 ml
and make up with water to 1000 ml.
Concentration of this solution is determined as follows:
Remove 10.00 ml; Add 25.00 ml of the standard solution of iodine (4.2.10)
and 10.00 ml of sodium hydroxide solution (4.2.7).
Leave to stand for 5 minutes.
Acidify with 11.00 ml of HCl (4.2.5) and titrate the excess iodine with a standard
Sodium Thiosulphate solution (4.2.11), as an indicator solution
starch (4.2.8).
Consumption of 1 ml of 0.05 mol/l standard solution of iodine (4.2.10) corresponds to 1.5
mg formaldehyde.
4.2.15 formaldehyde standard solution: dilute solution
Formaldehyde stock solution dilute at a ratio of 1/20 and then
in a ratio of 1/100. 1 ml of the resulting solution contains about 1 mikrog
formaldehyde.
The exact content is calculated.
4.3. Apparatus and equipment
4.3.1. Normal laboratory equipment
4.3.2 phase separation filter, Whatman 1 PS (or equivalent)
4.3.3 Centrifuge
4.3.4 water bath set at a temperature of 60 ° C
4.3.5 Spectrophotometer
4.3.6. Glass cells with optical path lengths of 1 cm
4.4 Procedure
4.4.1 the sample solution
In the 100 ml flask to the nearest 0.001 g weigh the quantity
the test sample (g) corresponding to a presumed quantity of formaldehyde
about 150 mikrog. Make up to the mark with water and mix (solution). (Is the
check that the pH is about 6; otherwise, add
hydrochloric acid solution (4.2.6).)
In an Erlenmeyer flask of 50 ml
-10.00 ml S,
-5.00 ml of the pentane-reagent (4.2.12),
-make up the final volume of the demineralized water 30 ml.
4.4.2 standard solution
Possible interference due to discoloration of the test sample is removed
the use of this reference solution:
In an Erlenmeyer flask of 50 ml
-10.00 ml S,
-5.00 ml reagent (4.2.13),
-demineralized water to a final volume of 30 ml.
4.4.3 the blank solution
In an Erlenmeyer flask of 50 ml, add:
-5.00 ml of the pentane-reagent (4.2.12),
-demineralized water to a final volume of 30 ml.
4.4.4 the determination
4.4.4.1 Mixtures prepared according points 4.4.1, 4.4.2 and 4.4.3.
A conical flask is exactly 10 minutes in a water bath heated
at 60 ° C Allow to cool 2. minutes in the bath with cold water.
4.4.4.2 content is converted into a 50-ml separating funnels containing 10.00 ml
butan1olu (4.2.4). Rinse each flask with 3 to 5 ml of water. The mixture is
thoroughly shake it for exactly 30 seconds. Allow the phases to separate.
4.4.4.3 Content is filtered into the measurement cells (4.3.2) through the filter for
the separation of the phases. You can also phase centrifuge (at 3000 rpm for 5
minutes).
4.4.4.4 At 410 nm of the absorbencies (A1) of the extract of the sample solution
(4.4.1) against the extract of the standard solution (4.4.2).
4.4.4.5 Similarly absorbencies (A2) the extract of the blank solution
you try to (4.4.3) against butan1olu.
Note: all the following operations must be carried out within 25 minutes from the
the moment when the Erlenmeyer flasks are placed in the bath at 60
St. (C).
4.4.5 calibration curve
4.4.5.1 in an Erlenmeyer flask of 50 ml, add:
-5.00 ml of the diluted standard solution as specified in point 4.2.15,
-5.00 ml of the pentane-reagent (4.2.12),
-demineralized water to a total volume of 30 ml.
4.4.5.2 proceed as described in 4.4.4 and measure the
the absorbance against butan1olu (4.2.4).
4.4.5.3 the Procedure with 10, 15, 20 and 25 ml of the diluted standard
solution (4.2.15).
4.4.5.4 the zero value (corresponding to the colour reagents)
gets the procedure laid down in point 4.4.4.5.
4.4.5.5 minus zero values from each absorbance values obtained
4.4.5.1 and 4.4.5.3 points according to the calibration curve. Beer
the law is valid up to 30 mikrog formaldehyde.
4.5 Calculations
4.5.1
From the values of A1, A2 and subtracts the value from the calibration curve (4.4.5.5)
(C) the quantity of formaldehyde in the sample solution (4.4.1) in mikrog.
4.5.2 the formaldehyde content of the sample (% m/m) is calculated using the formula:
(C)
formaldehyde content in% (m/m) =-------
103. m
where
m = mass of the test sample is in g.
4.6 the repeatability
For products containing about 0.2% (m/m) of formaldehyde the difference between the
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.005% for kolorimetrickou method with pentane, the nearest 0,001 g.
-Colorimetric determination of free formaldehyde the results
higher than the maximum permissible concentration under the
The Ministry of health no. 26/2001 Coll. on hygienic requirements
to cosmetic products, on the terms of the request for the omission
the ingredients of a cosmetic product on the packaging and on the requirements of the
education and the practice of natural persons responsible for the manufacture of cosmetic
the device and the evaluation of the safety of a cosmetic product for health
natural persons (cosmetic products), IE.
and up to 0.2 0.05%)% in the product, for which there is no formaldehyde content listed
on the label,
(b)) more than 0.2% in the product, stating the content of formaldehyde, or without
him,
the procedure must be applied in accordance with section 5.
5. The QUANTITATIVE DETERMINATION in the PRESENCE of FORMALDEHYDE DONORS
5.1 Principle
The separate formaldehyde is converted to the yellow derivative by reaction with lutidinu
pentane in post-column reactor and the derivative absorbance
shall be measured when 420 nm.
5.2 USED CHEMICALS
All the reagents must be of analytical purity or higher
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-(kept at 4 ° C)
5.2.5 DIPOTASSIUM hydrogen phosphate Anhydrous, sodium
5.2.6 orthophosphoric acid, 85% (d204 = 1.7)
5.2.7. Methanol, HPLC grade
5.2.8 Dichloromethane
5.2.9 formaldehyde 37% to 40% (m/V)
5.2.10. Sodium hydroxide, 1 mol/l
5.2.11. hydrochloric acid, 1 mol/l
5.2.12. hydrochloric acid, 0.002 mol/l
5.2.13 starch solution freshly prepared according to the European Pharmacopoeia
(see 4.2.8)
5.2.14 Standard iodine solution, 0.05 mol/l
5.2.15 standard Sodium Thiosulphate solution, 0.1 mol/l
5.2.16. mobile phase:
Aqueous solution of disodium sodium (5.2.5), 0.006 mol/l,
adjusted to pH 2.1 trihydrogenfosforečnou acid (5.2.6)
5.2.17 post-column Reagent into the reactor:
In a 1000 ml volumetric flask dissolve:
-62.5 g ammonium acetate (5.2.2),
-7.5 ml acetic acid (5.2.3),
-5 ml pentane, 4dionu (5.2.4).
Make up the volume with water (5.2.1).
The solution is kept in the dark.
Shelf life: maximum three days at 25 ° C.
The color of the solution should not change.
5.2.18 the formaldehyde standard solution: stock solution
10 g of formaldehyde (5.2.9) into a volumetric flask of 1000
ML and make up with water to 1000 ml.
Concentration of this solution is determined 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).
Leave to stand for 5 minutes.
Acidify with 11.00 ml of HCl (5.2.11) and titrate the excess iodine with a standard
Sodium Thiosulphate solution (5.2.15), as an indicator solution
starch (5.2.13).
Consumption of 1 ml of the standard iodine solution (5.2.14) is equivalent to 1.5 mg
formaldehyde.
5.2.19 the formaldehyde standard: diluted solution
Formaldehyde stock solution diluted in a ratio of 1/100 of the mobile phase
(5.2.16) 1 ml of the resulting solution contains about 37 mikrog formaldehyde.
The exact content is calculated.
5.3. Apparatus and equipment
5.3.1. Normal laboratory equipment
5.3.2 pulse-free pump for HPLC
5.3.3 Low-pressure pulse-free pump for the reagent (or a second
pump for HPLC)
5.3.4 Injection valve with a 10 mikrol
5.3.5
Post-column reactor with the following components:
+ three-neck flask, jednolitrová
+ 1 litre heater,
+ two Vigreux column column on at least 10 floors, air cooled,
+ stainless steel tube (used as a heat exchanger) 1.6 mm,
internal diameter 0.23 mm, length 400 mm
+ Teflon tube 1.6 mm, internal diameter 0.23 mm, length 5 m
(French-see annex 1),
+ one T-piece without any dead volume (Valco or equivalent),
+ three couplings without dead volume
or: one post-column module Applied Biosystems PCRS 520 or
equivalent to the reactor with a capacity of 1 ml.
5.3.6. membrane filter, pore size 0.45 mikrom
5.3.7 the Patron Saint SEPPAK (R) C18 or equivalent
5.3.8
The finished columns:
-Bischoff Hypersil RP 18 (type NC number C 25.46 1805),
(5 mikrom, length 250 mm, internal diameter = 4.6 mm),
-or DuPont, Zorbax ODS
(5 mikrom, length 250 mm, internal diameter = 4.6 mm),
-or Phase SEP, spherisorb ODS 2
(5 mikrom, length 250 mm, internal diameter 4.6 mm).
5.3.9 Gap
Bischoff K1 hypersil RP 18 (type K1 G 6301 1805)
(5 mikrom, length 10 mm) or equivalent.
5.3.10
Column, and the gap will combine the Ecotube system (type and 15,020,508 Bischoff)
or equivalent.
5.3.11
Apparatus (5.3.3) shall be drawn up according to the block diagram in annex 2.
Services for the nastřikovacím valve must be as short as possible. In this case,
It has a stainless-steel tube between the reactor outlet and the entry into the
the detector cool the mixture prior to detection; detector temperature is not known, but
is a constant.
5.3.12 detector for visible and UV range
– Writer
5.3.14 Centrifuge
5.3.15. ultrasonic bath
5.3.16 the vibrating stirrer (Vortex or equivalent)
5.4 the procedure
5.4.1. Calibration Curve
Construct the plotting peak heights as a function of the concentration of the diluted
formaldehyde calibration solutions.
Prepare the standard solutions by diluting the standard solution
formaldehyde (5.2.19) mobile phase (5.2.16):
-1.00 ml of solution (5.2.19) diluted to 20.00 ml (about 185 mikrog/100 ml)
-2.00 ml of solution (5.2.19) diluted to 20.00 ml (about 370 mikrog/100 ml)
-5.00 ml of solution (5.2.19) diluted to 25.00 ml (about 740 mikrog/100 ml)
-5.00 ml of solution (5.2.19) diluted to 20.00 ml (about 925 mikrog/100
ml).
Calibration solutions with about one hour left to stand at room
temperature and must be freshly prepared. The calibration curve is linear
for the concentration from 1.00 to 15.00 mikrog/ml.
5.4.2. Preparation of samples
5.4.2.1 Emulsions (creams, makeup, eyeshadow)
Into volumetric flasks, 100 ml, to the nearest 0.001 g
weigh the test sample (m grams) corresponding to a presumed quantity of 100
mikrog formaldehyde. Add exactly 20.00 ml Dichloromethane (5.2.8) and
20.00 ml hydrochloric acid (5.2.12). Mix using the
vibrating stirrer (5.3.16) and in an ultrasonic bath (5.3.15). Phase
separated by centrifugation (2 min at 3000 g). Meanwhile, with the Patron Saint
(5.3.7) 2 ml methanol (5.2.7), then condition with 5 ml water (5.2.1).
Pass a cartridge to 4 ml of the aqueous phase of the extract,
the first two millilitres is removed and the following fraction.
5.4.2.2 Skin lotions, shampoos
Into volumetric flasks, 100 ml, to the nearest 0.001 g weigh
of test sample (m grams) corresponding to a presumed quantity of 500 mikrog
formaldehyde.
Make up to the mark with mobile phase (5.2.16).
Filter the solution through a filter (5.3.6) and inject or leave
clean cartridge advance pass according to paragraph 5.4.2.1. All
solutions must be injected immediately after preparation.
5.4.3. Chromatographic conditions
-The flow rate of the mobile phase: 1.0 ml/min,
-Reagent Flow: 0.5 ml/min,
-The total flow rate of the output from the detector: 1.5 ml/min,
-Injection volume: 10 mikrol,
Elution temperature:-for difficult separations, immerse the column in a bath of ice
and wait for the temperature (15 to 20 minutes),
-Temperature of post-column reaction: 100 ° C,
-Detection: 420 nm.
Note:
The entire chromatographic system and post-column must be system after use
rinse with water (5.2.1). If the system does not apply to two days, must
After this rinsing to follow even rinsing with methanol (5.2.7).
Before running the new kondicionováním allow water to
prevent recrystallization.
5.5 Calculation
Emulsions: (5.4.2.1):
Formaldehyde content in% (m/m):
(C). 10-6. 100 (C). 10-4
--------------- = ----------
5 m 5 m
Skin lotions and shampoos:
In this case, apply the formula:
(C). 10-6. 100 (C). 10-4
--------------- = ----------
m m
where
m = mass of the sample analysed in g (5.4.2.1),
C = concentration of formaldehyde in 100 mikrog/ml deducted
from the calibration curve (5.4.1).
5.6 the repeatability
For products containing about 0.05% (m/m) of formaldehyde the difference between the
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.001%.
For products containing about 0.2% (m/m) of formaldehyde the difference between the
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.005%.
Annex 1
INSTRUCTIONS FOR MAKING A "FRENCH KNITTING"
NECESSARY ACCESSORIES
-Wooden bobbin:
external diameter 5 cm with a hole of diameter 1.5 cm in the middle. The coil is
affix four steel nails (figs. 7 and 8). The distance between the
two nails must be 1.8 cm and must be at a distance of 0.5 cm from the
the central hole;
-one fixed curved needle (hook) to the stretching of the Teflon tube;
-5 m Teflon tube 1.6 mm in diameter, with an internal diameter 0.3 mm.
How to:
Before the completion of a "French knitting" must be a Teflon capillary tube
drew central hole from the top end of the coil to the lower end of the (probably
10 cm of tube protruding from the bottom of the coil, which allows
stretch the chain during knitting); the tube is then wrapped around the four
nails, as shown in Figure 9.
The upper and lower part of the tissue will be protected by metal rings and cable
the screws; When fastening it is necessary to see to it that the teflon damage.
Tube wrapped around each nail for a second time, and in the following manner
creates a "stitch":
-the lower part of the tube over the upper part of the hook, pull tube
(Figure 10). This is gradually repeated on each nail (1, 2, 3 and 4
in anti-clockwise direction), until 5 m or the desired repeat
the length of the wire mesh.
It is necessary to leave around 10 cm of tube to close the chain. Tube
through each of the four loops and pull gently to the chain
He concluded.
Note: the French column reactors is commercially
available (Supelco).
Figure 7
Schematic representation of the spool
Figure 8
Figure 9
The first series
Figure 10
The second series
"Stitch" is created so that the bottom of the tube (indicated by a solid line)
drags through a second capillary tube (indicated by the shipping and receiving)
Figure 11
Annex 2
1 = HPLC pump
2 = injection valve
3 = column with pre-column
4 = reagent pump
5 = T-piece without dead volume
5 ' = T-piece (Vortex)
6 6 ' = clutch without dead volume
7 = "French knitting"
7 ' = reactor
8 = three-neck flask with boiling water
9 = heater
10 = condenser
11 = heat exchanger from stainless steel tube
' 11 ' = heat exchanger
12 = visible UV detector and area
13 = PCRS 520 post-column module
14. The QUANTITATIVE DETERMINATION of DICHLOROMETHANE and 1, 1, 1-TRICHLOROETHANE
1. scope and field of application
This method is described for the quantitative determination of dichloromethane
(methylene chloride) and 1, 1, 1-trichloroethane (methyl chloroform) in all
cosmetic products that could contain this solvent.
2. The DEFINITION of the
The contents of the dichloromethane and 1, 1, 1-trichloroethane in the sample as determined by the
method is expressed in percentage by mass.
3. The PRINCIPLE of the
This method uses gas chromatography with chloroform as internal
standard.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Chloroform (CHCl3)
4.2 carbon tetrachloride (CCl4)
Dichloromethane (CH2Cl2) 4.3
4.4 to 1, 1, 1-Trichloroethane (CH3CCl3)
4.5 the Acetone
4.6 the Nitrogen
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2. Gas Chromatograph fitted with katharometer detector.
5.3 converting the sample bottle to the 50 to 100 ml (see chapter "preparing
the samples in the laboratory ")
5.4 Pressure syringe, 25 or 50, mikrol
(see the chapter "preparing samples in the laboratory")
6. the procedure
6.1 Sample without overpressure:
weigh accurately a sample into a conical flask with stopper. Add
accurately weighed quantity of chloroform (4.1) as internal standard,
corresponding to the presumed quantity of dichloromethane and
1, 1, 1-trichloroethane contained in the sample. Mix thoroughly.
6.2 Sample in the aerosol dispenser:
Uses the sampling procedure, as described in the chapter "preparation of the sample in the
laboratory ", but with the following modifications:
6.2.1.
After converting the sample into the bottle (5.3) further into the bottle adds as an internal
standard volume of chloroform (4.1), corresponding to a presumed quantity of
dichloromethane and/or 1, 1, 1-trichloroethane contained in the sample.
Mix thoroughly. The dead volume of the valve with 0.5 ml
carbon tetrachloride (4.2). After drying, the difference shall specify
the added weight of internal standard.
6.2.2.
After filling the syringe with the pattern should be the tip of the syringe to rinse
with nitrogen (4.6), to her left before injection into the chromatograph
None of the rest of the sample.
6.2.3.
After you remove each sample should surface of the valve and the clutch several times
rinsed with acetone (4.5) (where necessary, uses injection
syringe) and then thoroughly dried with nitrogen (4.6).
6.2.4
Each analysis for the measurement of the cylinder shall be used for converting the two different
sample and for each bottle is made of five measurements.
7. CHROMATOGRAPHIC CONDITIONS
7.1 Gap
Column material: stainless steel.
Length: 300 mm.
Diameter: 3 or 6 mm.
Packing: same material as for the analytical column packing.
7.2 Column
The stationary phase is prepared from Hallcomidu M 18 on chromosorb. Column
must provide a resolution "R" equal to, or better than, 1.5, where:
(d) ' (r2-r1)
R = 2 --------------
W1 + W2
where:
R1 and r2 = retention times, two peaks (min.)
W1 and W2 = píkůu width at half height (in millimetres),
d ' = the chart speed (mm/min).
7.3.
Examples are given of the column, which provides the search results:
Column I II
Column material: stainless steel, stainless steel
Length: 350 cm 400 cm
Diameter: 3 mm 6 mm
Filling:
chromosorb: WAW-WAW DMCS-HP
grain size: 100 to 120 mesh 60-80 mesh
Stationary phase: Hallcomid M 18 Hallcomid M 18,,
10% 20%
Temperature conditions may vary depending on the device. In
the examples have been set as follows:
Column I II
Temperature:
column: 65 ° C 75 ° C
Injector: 150 ° C 125 ° C
detector: 150 ° C 200 ° C
Carrier gas:
helium flow rate: 45 ml/min to 60 ml/min
input pressure: 2.5 bar 2.0 bar
Injector: 15 mikrol 15 mikrol
8. MIX for the DETERMINATION of the RESPONSE FACTORS
Erlenmeyer flask with stopper, weigh accurately a mixture:
Dichloromethane (4.3), 30% (m/m),
1, 1, 1-trichloroethane (4.4), 35% (m/m),
Chloroform (4.1), 35% (m/m).
9. CALCULATIONS
9.1. the calculation of the coefficient of proportionality of the substance "p" due to "and"
selected as internal standard
If the first ingredient as "p", so it is
KP = its proportionality coefficient,
mp = its mass in the mixture,
AP = its peak area.
If the second substance as "and", so it is
Ka = its proportionality coefficient (= 1),
Ma = its mass in the mixture,
AA = its peak area,
then
mp x Aa
kp = ---------
Ma x Ap
For example, the following factors have been obtained (for the proportionality
chloroform: k = 1):
Dichloromethane: k1 = 0.78 +/-0.03
1, 1, 1-trichloroethane: k2 = 1.00 +/-0.03
9.2. Calculation of the% (m/m) of dichloromethane and 1, 1, 1-trichloroethane present in the
the sample to be analyzed
If:
Ma = mass of chloroform used (in g)
Ms = mass of the sample for analysis (in g)
AA = area of the chloroform peak,
A1 = peak area of dichloromethane,
A2 = peak area of 1, 1, 1-trichloroethane,
then
Ma x A1 x k1 x 100
% (m/m) CH2Cl2 = -----------------------
AA x Ms
Ma x A2 x k2 x 100
% (m/m) CH3Cl3 = -----------------------
AA x Ms
10. REPEATABILITY
For products containing approximately 25% (m/m) of dichloromethane and 1, 1, 1-
trichloroethane difference between the results of two parallel determinations on
the same sample should not exceed an absolute value of 2.5%.
15. The qualitative and QUANTITATIVE DETERMINATION of QUINOLIN-8-OL and
BIS (quinolin-8-OL)-SULFATE
1. scope and field of application
This method describes the qualitative and quantitative determination of
quinolin-8-OL and its sulphate.
2. The DEFINITION of the
Quinolin-8-OL and bis (quinolin-8-ol)-sulfate in the sample set
This method is expressed as percentage by weight chinolin8-OL.
3. The PRINCIPLE of the
3.1 qualitative determination
To the qualitative determination makes use of thin-layer chromatography.
3.2 quantitative determination
Quantitative determination is carried out by using the complex spectrophotometry
the resulting reaction with Fehling's solution at 410 nm.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1 the quinolin-8-ol
4.2 Benzene.
Due to its toxicity, it is necessary to work carefully with benzene.
4.3 Chloroform
4.4. Aqueous sodium hydroxide, 50% solution (m/m)
4.5 copper sulfate pentahydrate
4.6 draselnosodný Tartrate
4.7. hydrochloric acid, 1 m
4.8. sulphuric acid, 0, 5 m
4.9. sodium hydroxide solution, 1 m.
4.10 Ethanol
4.11 the Butan-1-ol
4.12. Glacial acetic acid
4.13. hydrochloric acid 0, 1 m
4.14 "Celite 545" or equivalent
4.15 standard solutions
4.15.1
In a 100 ml volumetric flask, weigh 100 mg of quinolin-8-OL (4.1).
Dissolve in a little sulphuric acid (4.8). Dilute acid
sulphuric acid (4.8) to the mark.
4.15.2.
In a 100 ml volumetric flask, weigh 100 mg of quinolin-8-OL. Dissolve
in ethanol (4.10). Make up to volume with ethanol (4.10) and mix thoroughly.
4.16 the fehling's solution
Solution And
In a 100 ml volumetric flask, weigh 7 g of copper II sulphate pentahydrate
(4.5). Dissolve in a small amount of water. Make up to the mark with water and
mix.
Solution B
In a 100 ml volumetric flask, weigh 35 g of draselnéhosodného
(2.9). Dissolve in 50 ml of water. Add 20 ml of sodium hydroxide
(4.4) make up to volume with water and mix. Immediately before use
into volumetric flasks, 100 ml pipette 10 ml of solution A and 10 ml
solution (B). Make up to the mark and mix.
4.17 eluting solvents for thin-layer chromatography
I: Butan-1-ol (4.11)/kyselina acetic acid (4.12)/water (80:20:20; V/V/V).
II: Chloroform (4.13)/kyselina acetic acid (4.12) (95:5; V/V).
4.18 the 2.6-Dichloro-4-(chloroin INO) cyclohexa-2,5 2.5-dienon, 1% (m/V) solution in
ethanol (4.10)
4.19. sodium carbonate, 1% (m/V) solution in water
4.20 Ethanol (4.10), 30% (V/V) solution in water
4.21 Disodium dihydrogen ethylenediaminetetraacetate--, 5% (m/V) solution in
water
4.22. Buffer solution, pH 7
Graduated flask, 1 litre, weigh 27 g anhydrous
potassium dihydrogen phosphate, and 70 g of diammonium phosphate
hydrogenorthophosphate trihydrate. Make up to the mark with water.
4.23 plates for thin-layer chromatography
The finished plates for thin-layer chromatography of thickness 0.25 mm
(eg. Merck Kieselgel 60 or equivalent). Before using the Board
spray 10 ml of reagent (4.21) and dried at 80 ° C.
5. apparatus and EQUIPMENT
flask with ground-glass joint on a 100 ml round-bottom
5.2. volumetric flask
5.3. Graduated pipettes, 10 and 5 ml
5.4. bulb pipettes, 20, 15, 10 and 5 ml
5.5. separating funnels, 100, 50 and 25 ml
5.6 the Pleated filter paper, diameter 90 mm
5.7. Rotary evaporator
5.8 the reflux condenser with ground
5.9 the Spectrophotometer
5.10 the cells with optical paths of length 10 mm
5.11 the Stirrer with heating
5.12 glass chromatography column dimensions:
160 mm long with a diameter of 8 mm, on the bottom, a small end of the stopper from the
glass wool, and with the application of pressure at the upper end.
6. the procedure
6.1. Qualitative determination
6.1.1 liquid samples
6.1.1.1. The section of the test sample is adjusted to pH 7.5 and 10 mikrol,
spotted on the starting line in advance of modified silica gel plates for
thin-layer chromatography (4.23).
6.1.1.2. In the other two points of the starting line apply 10 and 30 mikrol
standard solution (4.15.2), after the chromatogram has been developing in one of the
the developing solvent (4.17).
6.1.1.3 When his forehead forward about 150 mm, the plate is dried at 110 ° C (15
minutes). Under the UV lamp (366 nm) the quinolin-8-OL yellow fluoresce.
6.1.1.4 spray the plate with sodium carbonate solution (4.19). Dry
and spray with a solution of 2.6-Dichloro-4-(chloroin INO) cyclohexa-2,5 2.5-visualizing agent II
(4.18) the quinolin-8-OL turns blue.
6.1.2 solid samples or creams
6.1.2.1 1 g of the sample in 5 ml tlumivýho solution (4.22). Then
using 10 ml of chloroform (4.3) converts into a separating funnel and shake
After the separation of the chloroform layer the aqueous layer twice
extract 10 ml of chloroform (4.3). United and filtered
chloroform extracts in the round-bottomed flask at 100 ml (5.1) evaporated
almost to dryness on the Rotary evaporator (5.7). Dissolve the residue in 2 ml of
chloroform (4.3) and the 10 and 30 mikrol solution is applied to the
silica gel plate for thin-layer chromatography (4.23) in accordance with
the procedure described in point 6.1.1.1 and beyond.
6.1.2.2 deposit on a plate 10 and 30 mikrol standard solution (4.15.2)
and continue as described in 6.1.1.2 to 6.1.1.4.
6.2. The quantitative determination of
6.2.1 liquid samples
6.2.1.1 in the round-bottomed flask weigh 5 g of the sample. Add 1 ml of
sulphuric acid solution (4.8) and the mixture to evaporate almost to dryness under
reduced pressure at a temperature of 50 ° C.
6.2.1.2. Dissolve this residue in 20 ml of warm water. Converts to
volumetric flasks, 100 ml. Rinse three times with 20 ml of water. Make up to
100 ml of water and mix.
6.2.1.3 5 ml of this solution into a separating funnel on the pipette 50 ml
(5.5). Add 10 ml of fehling's solution (4.16). Copper complex
quinolin-8-OL is three times 8 ml of chloroform (4.3).
6.2.1.4 Chloroform layer though and gather in a volumetric flask
25 ml (5.2). Make up to the mark with chloroform (4.3) and shake.
The absorbance of the yellow solution is measured at 410 nm against chloroform.
6.2.2 solid samples and creams
6.2.2.1 To flask 100 ml round-bottomed (5.1) weigh 0.500 g of the
sample. Add 30 ml of benzene (4.2) and 20 ml of hydrochloric acid
(2.9). Solution with stirring for 30 minutes to boil under reflux.
6.2.2.2 content is converted into a 100 ml separating funnels (5.5). Rinse the
5 ml of 1 mol/l HCl (4.7). The aqueous phase is converted into the flask with round
on the day (5.1), wash the benzene phase with 5 ml of hydrochloric acid
(4.7).
6.2.2.3 in the case of emulsions that impede further progress, mix 0.500 g of the
the sample with 2 g of Celite 545 (4.14) to produce a free-flowing powder. The mixture is in the
small doses converts into a glass chromatography column (5.12).
After each addition, filling the column pushed down. When the whole pattern
into the column, elute with hydrochloric acid (4.13) in such a
in a way, that 10 ml of eluate in about 10 minutes (if it is a
necessary, the elution can be considered a slight overpressure of nitrogen). During the elution
ensure that the above column was still a little
hydrochloric acid. The first 10 ml of eluate is subjected to further
the procedure described in paragraph 6.2.2.4.
6.2.2.4 aqueous phases (6.2.2.2) or from the column eluate (6.2.2.3)
Rotary evaporator under reduced pressure evaporate almost to dryness.
6.2.2.5 dissolve the residue in 6 ml of sodium hydroxide solution (4.9).
Add 20 ml of fehling's solution (4.16) and the content is converted into a separating
a 50 ml separating funnel (5.5). Rinse the flask with 8 ml of chloroform (4.3).
Shake and filter the chloroform layer into a 50 ml volumetric flask
(5.2).
6.2.2.6 Extraction with 8 ml of chloroform (4.3) three times.
Chloroform phase is collected in a 50 ml volumetric flask. Make up
the mark with chloroform (4.3) and shake. Measure the absorbance of the yellow
solution at 410 nm against chloroform (4.3).
7. CALIBRATION CURVE
Into four 100 ml flasks round-bottomed (5.1), each of which contains 3
ml of 30% aqueous ethanol (4.20), pipette 5, 10, 15 and 20 ml
standard solution (4.15.1) corresponding to 5, 10, 15 and 20 mg
quinolin-8-OL. Proceed as described in section 6.2.1.
8. The CALCULATION of the
8.1 liquid samples
and
Quinolin-8-OL (in% (m/m)) =-x 100
m
where:
a = quantity of quinolin-8-OL, read from the calibration curve (7) in mg,
m = the mass (in milligrams) of the test portion (6.2.1.1).
8.2 solid samples or creams
2A
Quinolin-8-OL (in% (m/m)) =----x 100
m
where:
a = quantity of quinolin-8-OL, read from the calibration curve (7) in mg,
m = the mass (in milligrams) of the test portion (6.2.1.1).
9. REPEATABILITY
For products containing about 0.3% (m/m) the quinolin-8-OL, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.02%.
16. The QUANTITATIVE DETERMINATION of AMMONIA
1. scope and field of application
This method describes the determination of free ammonia in cosmetic
resource.
2. The DEFINITION of the
Ammonia content of the sample determined by this method is expressed in
percentage by mass of ammonia.
3. The PRINCIPLE of the
To a test sample of a cosmetic product diluted aqueous
methanol barium chloride solution is added. Any precipitate
filtered or centrifuged. This procedure prevents the loss of ammonia,
occur when the steam distillation apparatus for some ammonium
salts, such as the carbonate and bicarbonate and salts of fatty acids, with
the exception of ammonium acetate.
From the filtrate or supernatant to distil the ammonia with the steam, and
determine the potentiometric or other titration.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1 Methanol
4.2. Barium chloride dihydrate, 25% solution (m/V)
4.3 trihydrogenboritá Acid, 4% solution (m/V)
4.4. Sulphuric acid 0.25 mol/l standard solution
4.5 the Liquid odpěňovač
4.6. Sodium hydroxide, 0.5 mol/l standard solution
4.7. Indicator, as appropriate:
mix 5 ml of a 0.1% (m/V) methyl red in ethanol with 2 ml
0.1% solution (m/V) methylene blue solution in water.
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2. centrifuge with 100 ml tubes on the closable
5.3. Apparatus for steam distillation apparatus
5.4 Potentiometer
5.5. Glass electrode and a calomel electrode referentní.
6. the procedure
6.1
In a 100 ml volumetric flask, weigh a sample of (m)
corresponding to the maximum of 150 mg of ammonia.
6.2.
Add 10 ml of water, 10 ml of methanol (4.1) and 10 ml of the solution of chloride
barium (4.2). Make up to 100 ml with methanol (4.1).
6.3.
Mix and allow to stand overnight in the refrigerator (5 ° C).
6.4.
Then cool the solution filtered or centrifuged for 10 minutes in a closed
cells, in order to obtain a clear filtrate or supernatant layer.
6.5.
40 ml of the clear solution with pipette into the distillation
water vapour (5.3) and possibly add 0.5 ml of liquid Protein skimmers
(4.5).
4.1
Distil and collect 200 ml of distillate in a 250 ml beaker containing
10 ml of standard sulphuric acid (4.4) and 0.1 ml of indicator
(4.7).
4.2
Titrate the excess of acid with a backward-sodium hydroxide standard solution
(4.6).
4.2
Note:
For potentiometric determination, collect 200 ml of distillate in the beaker to
250 ml containing 25 ml of orthoboric acid solution (4.3) and
Titrate with standard sulphuric acid solution (4.4),
neutralization curve.
7. CALCULATIONS
7.1 calculation in the case of back titration
If the
V1 = the volume of the used sodium hydroxide solution (4.6)
(ml),
M1 = the actual concentration of sodium hydroxide solution (4.6),
M2 = the actual concentration of the sulphuric acid solution (4.4),
m = mass of the collected the test sample (6.1), (in mg)
then:
(20 M2-V1M1) x 17 x 100 (20 M2-V1M1) x 4250
%amoniaku (m/m) = -------------------------- = ----------------------
0.4 m
7.2 Calculation in the case of direct potentiometric titration
If the
V2 = the volume of the used sulphuric acid solution (4.4) (ml),
M2 = the actual concentration of the sulphuric acid solution (4.4),
m = mass of the collected the test sample (6.1), (in mg)
then:
V2 x M2 x 17 x 100 4250 V2 M2
amoniak = (m/m) = ------------------- = ------------
0.4 m
8. REPEATABILITY
For products containing about 6% (m/m) the difference between the ammonia
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.6%.
17. The qualitative and quantitative DETERMINATION of NITROMETHANE
1. scope and field of application
This method is suitable for qualitative and quantitative determination of
Nitromethane to the content of about 0.3% in cosmetic products in the
aerosol packaging.
2. The DEFINITION of the
The content of Nitromethane in the sample determined by this method is expressed in
percentage of nitromethane, calculated with reference to the total content
of the water resource.
3. The ESSENCE of the METHOD
Nitromethane is qualitatively determined on the basis of a colour reaction.
Nitromethane is quantitatively determined by gas chromatography after adding
the internal standard.
4. QUALITATIVE DETERMINATION
4.1 USED CHEMICALS
All the reagents must be of analytical purity or higher
4.1.1. Sodium hydroxide, 0.5 mol/l solution
4.1.2 Folin's reagent
Dissolve 0.1 g of 3.4-dihydro-3.4-dioxonaftalen-1-sulfonate
sodium and make up to 100 ml.
4.2 Procedure
To 1 ml of the sample, add 10 ml of solution 1 ml of 4.1.1 and 4.1.2.
A violet coloration indicates the presence of Nitromethane.
5. QUANTITATIVE DETERMINATION of
5.1 USED CHEMICALS
All the reagents must be of analytical purity or higher
5.1.1. Chloroform (internal standard 1)
5.1.2 2.3-Dimethylheptane: (internal standard 2)
5.1.3. Ethanol, 95%
5.1.4 the Nitromethane
5.1.5 chloroform reference solution
Into a tared 25 ml volumetric flask and add about 650 mg of chloroform
(5.1.1). The flask and contents are precisely weighed again. Make up to 25 ml of 95%
ethanol (5.1.3). Weigh and calculate the percentage by weight
chloroform in this solution.
5.1.6 2.4-dimethylheptane reference solution
Prepare similarly to chloroform reference solution but weigh
270 mg of 2.4-dimethylheptane (5.1.2) into a 25 ml volumetric flask.
5.2. Apparatus and equipment
5.2.1. Gas Chromatograph, with flame ionization detector
5.2.2. Equipment for sampling of aerosols
(the sample bottle, microsyringe connectors, etc.), how they are
described in the chapter "preparation of samples in the laboratory".
5.2.3. Normal laboratory equipment
5.3 How to
5.3.1. Preparation of the sample
Into a tared transfer bottle 100 ml sample, flushed out or
evacuated according to the procedure described in point 5.4 of chapter "preparation of the sample
in the laboratory ", add about 5 ml of one of the solutions of the internal standards
(5.1.5 or 5.1.6). Apply 10 ml or 20 ml glass syringe without
needle adapted to connect to the connector in accordance with the procedure described in point 5
the chapter "preparation of samples in the laboratory". Reweigh to
fixed the amount added. The same procedure is in this bottle converts
about 50 g of water and the content of the resource. Reweigh to
fixed the amount of the converted sample. Mix well.
Inject about 10 mikrol using special microsyringe (5.2.2).
This is done five times.
5.3.2. Preparation of the standard
Into volumetric flasks, 50 ml, weigh about 500 mg of Nitromethane exactly
(5.1.4) and either 500 mg of chloroform (5.1.1) or 210 mg of 2.4-
dimethylheptane (5.1.2). Make up to a volume of 95% ethanol (5.1.3).
Mix well. 5 ml of this solution into the volumetric flask to the
20 ml. Make up to a volume of 95% ethanol (5.1.3).
Inject about 10 mikrol using special microsyringe (5.2.2).
This is done five times.
5.3.3 conditions for gas chromatography
5.3.3.1 Column
It consists of two parts; the first contains as didecyl phthalate on
Gas chrom Q, the second HAVING UCON 50 HB 280 x on Gas Chrom Q. Ready
the combined column must yield a resolution "R" equal to, or better
than 1.5, where:
(d) ' (r2-r1)
R = --------------
W1 + W2
where
R1 and r2 = retention times, in minutes, of two of the peaks,
W1 and W2 = peak width at half height (in millimetres),
d ' = the chart speed (mm/min).
Column And:
Material: stainless steel.
Length: 1.5 m.
Diameter: 3 mm.
Filling: 20% didecyl phthalate on Gas chrom Q (100 to 120 mesh).
Column (B):
Material: stainless steel.
Length: 1.5 m.
Diameter: 3 mm.
Filling: 20% UCON 50 HB 280 x on Gas Chrom Q (100 to 120 mesh).
5.3.3.2 Detector
A suitable sensitivity setting elektrometru plamenového ionization
the detector is 8 × 10-10A.
5.3.3.3 temperature conditions
As appropriate, the following conditions have been shown to: injector: 150 ° C
Detector: 150 ° C column: between 50 and 80 ° C, depending on the
used columns and apparatus.
5.3.3.4 Gases
Carrier gas: nitrogen. Pressure: 2.1 bar. Flow: 40 ml/min for Gases
detector: the detector according to the manufacturer's specifications.
6. The CALCULATIONS
6.1. The proportionality factor of nitromethane, used to
the internal standard
If the Nitromethane as "n",
While
kN = its proportionality coefficient,
m ' n = its mass in the mixture (in grams),
S ' n = its peak area,
and if the internal standard, chloroform or
2-dimathylheptane: such as "c",
While
m'c = its mass in the mixture (in g)
' (C) = its peak area,
then:
m ' n S ' (c)
kn = ----- x --------
m ' c ' n
(kn is a function of the apparatus).
6.2 concentration of Nitromethane in the sample
If the Nitromethane as "n",
While
kN = its proportionality coefficient,
S ' n = its peak area,
and if the internal standard, chloroform or 2.4-
dimethylheptane: such as "c",
While
MC = its mass in the mixture (in g)
SC = its peak area,
M = the mass of the transferred resource dispenser (in g)
then the% (m/m) Nitromethane in the sample is
MC CN x Sn
-x-------x 100
M Sc
7. REPEATABILITY
For products containing about 0.3% (m/m) nitromethane, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.03%.
18. The qualitative and QUANTITATIVE DETERMINATION of MERCAPTOACETIC ACID
(SULFANYLOCTOVÉ) IN PERMS, STRAIGHTENING AND
RESOURCES FOR HAIR REMOVAL
1. scope and field of application
This method describes the qualitative and quantitative determination of
mercaptoacetic acid in perms, straightening
the hair and the hair removal.
2. The DEFINITION of the
Mercaptoacetic acid content of the sample determined by this method is
expressed in percentage by mass of mercaptoacetic acid.
3. The PRINCIPLE of the
Qualitative determination of mercaptoacetic acid is carried out either by color
response, or by using thin-layer chromatography and quantitative
determined by iodometry or gas chromatography.
4. QUALITATIVE DETERMINATION
4.1 qualitative determination of colour reaction
4.1.1 USED CHEMICALS
All the reagents must be of analytical purity or higher
4.1.1.1 lead di(acetate) paper
4.1.1.2. hydrochloric acid solution (1:1)
4.1.2 How to
4.1.2.1 qualitative determination of mercaptoacetic acid by using color
the reaction with the test lead acetate paper
A drop of sample is placed on the lead di(acetate) paper (4.1.1.1). If
the intense yellow colour appears, mercaptoacetic acid
probably present.
Sensitivity: 0.5%.
4.1.2.2 Qualitative determination of inorganic sulfides formation of hydrogen sulphide
After acidification
In the test tube, converts several milligrams of the sample analysed. Add
with 2 ml of distilled water and 1 ml of hydrochloric acid (4.1.1.2).
Released hydrogen sulfide which can be detected by smell, and on the paper with the
lead acetate paper (4.1.1.1) black lead sulphide precipitate is formed.
Sensitivity: 50 ppm.
4.1.2.3 qualitative determination of sulphites, sulphur dioxide, after the emergence of the
acidification
Proceed according to section 4.1.2.2. Bring to a boil. Sulphur dioxide
can be recognizable by its smell and its ability to reduce, for example,
manganisté ions.
4.2 qualitative determination by thin layer chromatography
4.2.1 USED CHEMICALS
Unless stated otherwise, the chemicals used must be of analytical purity or
higher
4.2.1.1 mercaptoacetic acid, 98% minimum purity,
by iodometry
4.2.1.2, 2 '-dithiodi (acetic acid), 99% minimum purity,
determined by iodometry
4.2.1.3 Acid 2-merkaptopropionová (Thiolactic acid), minimum
the purity of 95%, determined by iodometry
4.2.1.4 Acid 3-merkaptopropionová, 98% minimum purity,
determined by iodometry
4.2.1.5 3-merkaptopropan-1.2-diol (1-thioglycerol), minimum purity
98%, determined by iodometry
4.2.1.6 Finished silica gel plates for thin-layer chromatography of
thickness 0.25 mm
4.2.1.7 Finished plates for thin-layer chromatography with carbon
alumina, Merck F 254 E or equivalent
4.2.1.8, concentrated hydrochloric acid (d204 = 1.19 g/ml)
4.2.1.9 Ethyl acetate
4.2.1.10 Chloroform
4.2.1.11 diisopropyl ether
4.2.1.12 carbon tetrachloride
4.2.1.13. acetic acid
4.2.1.14. potassium iodide solution, 1% (m/V) in water
platičitý Chloride (4.2.1.15), 0.1% solution (m/V) in water
4.2.1.16 mobile phase
4.2.1.16.1 Ethyl acetate (4.2.1.9), chloroform (4.2.1.10),
diisopropyl ether (4.2.1.11), acetic acid (4.2.1.13) (20:20:10:
10, V/V/V/V)
4.2.1.16.2 Chloroform (4.2.1.10), acetic acid (4.2.1.13) (90:20,
IN/IN)
4.2.1.17 Detection reagents
4.2.1.17.1 immediately before use, mix equal volumes of solution
4.2.1.14 and solution (4.2.1.15).
4.2.1.17.2 bromine solution, 5% (m/V): dissolve 5 g of bromine in 100 ml
carbon tetrachloride (4.2.1.12).
4.2.1.17.3, 0.1% (m/V): 100 mg of fluorescein is
dissolved in 100 ml of ethanol.
4.2.1.17.4 Heptamolybdenan hexaamonný, 10% solution (m/V) in water.
4.2.1.18 standard solutions
4.2.1.18.1 thiglykolová Acid (4.2.1.1), 0.4% (m/V) in water.
4.2.1.18.2 2, 2 '-dithiodi (acetic acid) (4.2.1.2), 0.4% (m/V)
in the water.
4.2.1.18.3 2-merkaptopropionová Acid (4.2.1.3), 0.4% (m/V)
in the water.
4.2.1.18.4 3-merkaptopropionová Acid (4.2.1.4), 0.4% (m/V)
in the water.
4.2.1.18.5.3-merkaptopropan-1.2-diol (4.2.1.5), 0.4% (m/V)
the water.
4.2.2 apparatus
Normal equipment for thin-layer chromatography
4.2.3 Procedure
4.2.3.1. Preparation of samples
The sample is acidified with a few drops of hydrochloric acid (4.2.1.8) on
Ph 1 and filter if necessary.
In some cases it may be advisable to dilute the sample. In this case, the
acidification with hydrochloric acid performs before dilution.
4.2.3.2 Developing
Deposit on a plate 1 mikrol sample solution (4.2.3.1) and 1, mikrol each
of the five reference solutions (4.2.1.18). Carefully drain in temperate
a nitrogen stream and solvents (4.2.1.16.1 or
4.2.1.16.2). The plate is dried under nitrogen as quickly as possible, in order to avoid
the oxidation of thiols.
4.2.3.3 Detection
Spray the plate with one of the three reagents (4.2.1.17.1, 4.2.1.17.3 or
4.2.1.17.4). If the spray reagent (4.2.1.17.3),
bromine vapour (e.g. in a container containing a small beaker of reagent
(4.2.1.17.2)), until the stains are visible. Detection of the spray reagent
(4.2.1.17.4) will be successful only if the drying time shall not exceed 30
minutes.
4.2.3.4 Interpretation
The Rf values and colours of the reference solutions are compared with samples of the solutions.
The average Rf values given below are used only as a rough guide for
the comparison. Depends on:
. the level of activation of the thin layer at the time of chromatographing,
. the temperature in the tank Chamber.
Examples of Rf values obtained on a silica gel layer
--------------------------------------------------
Eluting solvents
-----------------------
4.2.1.16.1 4.2.1.16.2
--------------------------------------------------
Mercaptoacetic acid 0.25 0.80
--------------------------------------------------
Acid 0.40 0.95
2-merkaptopropionová
--------------------------------------------------
2, 2 '-dithiodi (acetic 0.00 0.35
acid)
--------------------------------------------------
Acid 0.45 0.95
3-merkaptopropionová
--------------------------------------------------
3-merkaptopropan-1.2-diol 0.45 0.35
--------------------------------------------------
5. QUANTITATIVE DETERMINATION of
(Determination of mercaptoacetic acid is carried out with the unused
the product of a freshly opened the packaging, in order to avoid oxidation.)
The determination must always begin with the iodometric procedure.
5.1 Jodometrie
5.1.1 Principle
The determination is carried out by the group "-SH" group with iodine in an acid medium
According to the equation:
2 + CH2SH HOOC-I2--> (HOOC-CH2-S) 2 + 2I-+ 2 h +
5.1.2 USED CHEMICALS
Iodine, 0.05 mol/l standard solution
5.1.3 apparatus
Usual laboratory equipment
5.1.4 Procedure
In an Erlenmeyer flask on the 150 ml, containing 50 ml
distilled water, accurately weigh 1 g of the sample to 0.5. Add 5 ml of
hydrochloric acid (4.1.1.2) (pH of solution about 0) and Titrate
iodine solution (5.1.2) until the yellow colour. You can use the
also the indicator (e.g. starch solution or carbon tetrachloride).
5.1.5. The calculation of the
Mercaptoacetic acid content is calculated according to the formula:
92 x n x 100 0.92 n
% (m/m) = -------------- = -------
x 10 x 1000 m m
where:
m = mass of the test sample (g)
n = the volume consumed by the iodine solution (5.1.2).
5.1.6 Notes
If the calculated result for mercaptoacetic acid is at least 0.1%
less than the maximum allowable concentration is not another determination necessary.
If the result is equal to or higher than the allowed maximum
concentration and qualitative determination of presence revealed several
reducing agents, it is necessary to perform the determination of gas
chromatography.
5.2. Gas Chromatography
5.2.1 Principle
Mercaptoacetic acid is separated from the auxiliary substances by precipitation of solution
cadmium di(acetate). After the diazomethanem methylation, which either prepare
in situ or in advance as a solution in diethyl ether, methylderivát
mercaptoacetic acid gas/liquid chromatography provides s
methyl-oktanoátem as internal standard.
5.2.2 the REAGENTS USED
All the reagents must be of analytical purity or higher
5.2.2.1 mercaptoacetic acid, 98%
5.2.2.2. hydrochloric acid (d204 = 1.19 g/ml)
5.2.2.3. Methanol
5.2.2.4 cadmium Acetate dihydrate, 10% solution (m/V) in water
5.2.2.5 Methyl octanoate, 2% solution (m/V) in methanol
5.2.2.6 Acetate buffer solution (pH 5):
Sodium acetate trihydrate, 77, g.
Acetic acid (glacial), 27.5 g.
Demineralized water to a final volume of one litre.
5.2.2.7. hydrochloric acid, freshly prepared 3 m solution in
methanol (5.2.2.3)
5.2.2.8 1-methyl-3-nitro-1-nitrosoguanidin
5.2.2.9 sodium hydroxide, 0,5 m solution
5.2.2.10 Iodine, 0,05 m standard solution
5.2.2.11. diethyl ether
5.2.2.12 diazomethane Solution prepared from the NmethylNnitrosotoluen-
4sulfonamidu (Fieser, Reagents for Organic Synthesis (Wiley), 1967)
The solution obtained contains about 1.5 g of diazomethane in 100 ml of diethyl ether.
Since diazomethane is a toxic and very unstable gas, it is necessary to
all the operations carried out in a fume cupboard, and stretching is a must
avoid the use of the zábrusové apparatus (for this purpose are available
special kits).
5.2.3 apparatus
5.2.3.1. Normal laboratory equipment
5.2.3.2. The apparatus for the preparation of diazomethane for in situ methylation (see
Fales, H.M., T.M. and Babashak, H.m., J.F., the analyte. Chem., 1973, 45,
2302)
5.2.3.3. The unit for the preparation of diazomethane (fieser)
5.2.4. Preparation of the sample
Into a 50 ml centrifuge tube accurately weigh enough sample to
It contained the anticipated quantity of 50 to 70 mg of mercaptoacetic acid.
Acidify with a few drops of hydrochloric acid (5.2.2.2) to
achieve a pH of about 3.
Add 5 ml of demineralized water and 10 ml of acetate tlumivýho
solution (5.2.2.6).
By using pH paper verifies that the pH value is about 5. Then add 5 ml of
of cadmium di(acetate) solution (5.2.2.4).
Wait 10 minutes and then centrifuge for at least 15 minutes at 4000 g.
Remove the supernatant liquid which may contain an insoluble fat (in the
the case of cream). This fat cannot be confused with the thiols,
which accumulate as a compact mass at the bottom of the container. Verifies that the
After adding a few drops of cadmium di(acetate) solution (5.2.2.4) to
supernatant.
If the previous did not reveal the presence of other qualitative determination
reducing agents than the thiols, check iodometrically that the quantity
thiolu present in the supernatant liquid does not exceed 6 to 8% of the original
the quantity.
Into the centrifuge tube containing the precipitate with 10 ml of methanol
(5.2.2.3) and finely dispersed precipitate rod. Centrifuge again for
at least 15 minutes at 4000 g. decant the Supernatant liquid and verify that
thiols.
The precipitate is washed with the same procedure again.
To the same centrifuge tube, add
. 2 ml of the methyl-oktanoátu (5.2.2.5),
. 5 ml of hydrochloric acid in methanol (5.2.2.7).
Completely dissolve the thiols (may only have a small amount of insoluble
the rest of the excipients). This is the solution "S".
The aliquot of this solution iodometrically verifies that the contents of the
thiols are at least 90% of the value obtained in point 5.1.
5.2.5 Methylation
Methylation is carried out either by using in situ preparation (5.2.5.1) or in advance
prepared diazomethane solution (5.2.5.2).
5.2.5.1 Methylation in situ
Into the methylation apparatus (5.2.3.2) containing 1 ml of diethyl ether
(5.2.2.11) 50 mikrol of solution "S" and methylate by the procedure
(5.2.3.2) with about 300 mg of 1-methyl-3-nitro-1-nitrosoguanidinu (5.2.2.8).
After 15 minutes (the ether solution should be yellow, which indicates a
the excess diazomethane) sample solution is transferred to the vial 2 ml
airtight stopper. Leave in the refrigerator overnight.
Methylate two samples at the same time.
5.2.5.2 methylation of previously prepared diazomethane solution
(D)
about a 5 ml stoppered transfer 1 ml of diazomethane solution
(5.2.2.12) then 50 mikrol of solution "S". Leave in the fridge over
the night.
5.2.6 Preparation standard
Prepare a standard solution of mercaptoacetic acid (5.2.2.1) of known
concentration, containing about 60 mg of pure mercaptoacetic acid
(5.2.2.1) in 2 ml.
This solution is "E".
Precipitate, assay and methylate as described in points
5.2.4 and 5.2.5.
5.2.7 the conditions for gas chromatography
5.2.7.1 Column
Type: stainless steel.
Duration: 2 m.
Diameter: 3 mm.
5.2.7.2 Refill
20% didecyl phthalate/Chromosorb, WAW 80 to 100 mesh.
5.2.7.3 Detector
Flame ionization. A suitable sensitivity setting elektrometru
plamenového ionization detector is 8 × 10-10 a.
5.2.7.4 Gases
Carrier gas: nitrogen.
pressure: 2.2 bar,
flow: 35 ml/min.
Auxiliary gas: hydrogen.
pressure: 1.8 bar,
flow: 15 ml/min.
Detector supplies: as specified by the instrument manufacturer.
5.2.7.5 temperature conditions
Injector: 200 ° C
Detector: 200 ° C
Column: 90 ° C.
5.2.7.6 speed recorder
5 mm/min.
5.2.7.7 quantity
3 mikrol. Carry out five injections.
5.2.7.8 chromatographic conditions are given as a guide only.
To reach a resolution "R" equal to, or better than, 1.5, where:
(d) ' (r2-r1)
R = 2 -------------
W1 + W2
where
R1 and r2 = retention times, two peaks (min.)
W1 and W2 = peak width at half height (in millimetres),
d ' = the chart speed (mm/min).
It is recommended to increase the temperature of the chromatography measurement of 90
St. (C) 150 ° C at 10 ° C per minute to eliminate
substances which might interfere with subsequent measurements.
5.2.8 Calculations
coefficient the coefficient of proportionality for mercaptoacetic acid
Calculated relative to methyl octanoate on the basis of the standard mixture.
If the mercaptoacetic acid as "t",
While
KT = its proportionality coefficient,
m't = its mass in the mixture (in mg)
With 't = area of the peak,
and if the methyloktanoát as "c",
While
m'c = its mass in the mixture (in mg)
' (C) = its peak area,
then
m 't ' (c)
K1 =-x-
m ' c 't
This coefficient varies according to the used equipment.
5.2.8.2 Concentration of mercaptoacetic acid present in the sample
If the mercaptoacetic acid as "t",
While
KT = its proportionality coefficient,
St = its peak area,
and if the methyloktanoát as "c",
While
MC = its mass in the mixture (in mg)
SC = its peak area,
M = the mass of the initial sample test (in mg)
then the% (m/m) mercaptoacetic acid present in the sample is:
MC kt x St
-x-------x 100
M Sc
8. REPEATABILITY
For products containing about 8% (m/m) mercaptoacetic acid must not
the difference between the results of two parallel determinations on the same sample
shall not exceed an absolute value of 0.8%.
19. The qualitative and QUANTITATIVE DETERMINATION of HEXACHLOROPHENE (INN)
AND QUALITATIVE DETERMINATION
1. scope and field of application
This method is suitable for all cosmetic products.
2. The PRINCIPLE of the
Hexachlorophene in the sample is extracted with ethyl acetate and qualitatively is
be determined by thin layer chromatography.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 the sulphuric acid 4 mol/l
3.2. Celite AW
3.3. Ethyl acetate
3.4. mobile phase: Benzene containing 1% (V/V) glacial acetic acid.
3.5 the visualizing agent i:
Rhodamine B: dissolve 100 mg of rhodamine B in a mixture of 150 ml
diethyl ether, 70 ml of absolute ethanol and 16 ml of water.
3.6 the visualizing agent II:
Solution of 2.6-dibromo-4-(chloroin INO) cyclohexa-2,5 2.5-visualizing agent II: 400 mg of 2.6-
dibromo-4-(chloroin INO) cyclohexa-2,5 2.5-visualizing agent II is dissolved in 100 ml
methanol (prepared fresh daily).
Sodium carbonate solution: 10 g of sodium carbonate is dissolved in 100
ml of demineralized water.
3.7. standard solution:
Hexachlorophene, 0.05% solution (m/V) in ethyl acetate
4. apparatus and EQUIPMENT
4.1 TLC plates, Kieselgel 254 200 x 200 mm (or equivalent)
4.2 the usual TLC equipment
4.3 Bath for conditioning the chromatographic Chamber maintained at a temperature of 26
St. C
5. preparation of the TEST SAMPLE
5.1
1 g of homogenized sample is thoroughly mixed with 1 g of Celite AW (3.2)
and 1 ml of sulphuric acid (3.1).
5.2.
Dry 2 hours at 100 ° C.
5.3.
Allow to cool and dry the rest of the crushed to a fine powder.
5.4.
Extract twice with 10 ml of ethyl acetate (3.3), after each extraction
centrifuge and combine the ethyl acetate layers.
5.5
Evaporate at 60 ° C.
5.6
Dissolve the residue in 2 ml of ethyl acetate (3.3).
6. the procedure
6.1
2 mikrol of the test sample solution (5.6) and 2 mikrol standard
solution (3.7) with the TLC plate for thin-layer chromatography
(4.1).
6.2.
Chamber (4.3) with the mobile phase (3.4).
6.3.
Plate for thin-layer chromatography is inserted into the Chamber and develops
to a distance of 150 mm of the forehead.
6.4.
Plate for thin-layer chromatography is removed from the tank and dry in
oven with forced circulation of air at a temperature of about 105 ° C.
6.5 Detection
Hexachlorophene spots on the plate for thin-layer chromatography,
detect in the manner described in section 6.5.1 or 6.5.2.
6.5.1
The plate evenly the visualizing agent I (3.5). After 30 minutes of
the plate under UV light at 254 nm.
6.5.2
Spray evenly on the plate with a solution of 2,6 dibromo-
4 (chloroin INO) cyclohexa-2,5 2.5-detection reagent II visualizing agent II (3.6).
Then spray with a solution of sodium carbonate (3.6). The plate is
After 10 minutes of watching the drying at room temperature, in the light of day.
7. INTERPRETATION
7.1 the visualizing agent I (3.5):
Hexachlorophene is detected as a bluish spot on a yellow-orange
fluorescent background and has an Rf of approximately 0.5.
7.2 the visualizing agent II (3.6):
Hexachlorophene is detected as a sky-blue to turquoise coloured
a stain on a white background and has an Rf of approximately 0.5.
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
This method is suitable for all cosmetic products.
2. The DEFINITION of the
The content of hexachlorophene in the sample determined by this method is expressed in
percentage by mass of hexachlorophene.
3. The PRINCIPLE of the
Hexachlorophene is determined after conversion to methylderivát gas
chromatography with electron capture detection.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Ethyl acetate
4.2 the N-methyl-N-nitroso-p-toluenesulphonamide by (diazald)
4.3. diethyl ether
4.4 Methanol
4.5 2-(2-ethoxyethoxy) ethanol (karbitol)
4.6 the formic acid
4.7. potassium hydroxide, aqueous solution of 50% (m/m) (always prepare a
fresh)
4.8 the Hexane for spectroscopy
4.9 Bromochlorophene (standard no. 1)
4.10 4.4 ', 6.6 '-tetrachlor-2, 2 '-thiodiphenol (standard no 2)
4.11 the 2, 4, 4 '-trichloro-2-hydroxydifenylether (standard no. 3)
4.12 the Acetone
4.13. sulphuric acid, 4 m
4.14 Celite AW
4.15 in ethyl acetate, formic acid, 10% solution (in/in)
4.16 Hexachlorophene
5. apparatus and EQUIPMENT
5.1. Usual laboratory glassware
5.2 Mini-apparatus for the preparation of diazomethane (Analyte. Chem., 1973, 45,
23022)
5.3 the gas chromatograph equipped with an electron capture detector is
63Ni source
6. the procedure
6.1 preparation of standard solution
The standard is chosen so that neinterferoval any substance contained in the
the analysis of the product as excipient. Usually it is best to
Standard No. 1 (4.9).
6.1.1.
In a 100 ml volumetric flask, weigh about 50 mg of standard no 1, 2 or 3
exactly (4.9, 4.10 or 4.11) and 50 mg of hexachlorophene (4.16). Make up
on the volume of ethyl acetate (4.1) (solution A). 10 ml of the solution and dilute
ethyl acetate (4.1) on the 100 ml (solution B).
6.1.2.
In a 100 ml volumetric flask, weigh about 50 mg of standard no 1, 2 or 3
exactly (4.9, 4.10 or 4.11). Make up to volume with ethyl acetate (4.1)
(solution C).
6.2. Preparation of the sample
Weigh approximately 1 g of homogenized sample and mix thoroughly
with 1 ml of sulphuric acid (4.12), 15 ml of acetone (4.12) and 8 g of Celite AW
(4.14). dry the mixture for 30 minutes in the air in a water bath, then one and
half an hour to dry in an oven with forced circulation of the air. Allow to
cool down, the rest is crushed to a fine powder and converts into a glass
the column. Elute with ethyl acetate (4.1) and collect 100 ml. Add 2 ml
internal standard solution (solution C) (6.1.2).
(Whereas, hexachlorophene is contained in a wide variety of product types,
It is important to lay down before the final recording of the results of the proceeds
in the determination of hexachlorophene in the sample by this procedure. If they are
low yields, can be made with the consent of the parties involved
changes, such as changing the solvents (benzene instead of ethyl acetate), etc.)
6.3 the methylation of the sample
All reagents and apparatus 2 hours cooling to a temperature of 0 to
4 ° C. To the outside of the apparatus diazomethanové stack converts 1.2
ml of the solution obtained in accordance with section 6.2 and 0.1 ml of methanol (4.4). To
the middle of the stack, converts about 200 mg of diazald (4.2), add 1 ml of
of carbitol (4.5) and 1 ml of diethyl ether (4.3) and dissolve.
Apparatus, half immersed in the bath at a temperature of 0 ° C and
in the middle of the stack with a syringe about 1 ml
cooled potassium hydroxide solution (4.7). It is necessary to
make sure that the yellow coloring diazomethane
persists. If the yellow colour does not persist, repeat the methylation with a
a further 200 mg of diazald (4.2).
(The persistence of this yellow coloring is diazomethane,
which is necessary for complete methylation of the sample.)
After 15 minutes the apparatus removed from the bath and leave closed when
room temperature for 12 hours. The apparatus is open, the excess
diazomethane by the addition of a few drops of a 10% (V/V) solution
formic acid in ethyl acetate (4.15), an organic solution to
25 ml volumetric flask and make up to volume. hexane (4.8).
1.5 mikrol this solution is injected into the chromatograph.
6.4 the methylation of the standard
All reagents and apparatus 2 hours cooling to a temperature of 0 to
4 ° C. Into the external tank of the apparatus is placed diazomethanové:
0.2 ml of solution B (6.1.1)
1 ml of ethyl acetate (4.1),
0.1 ml of methanol (4.4).
In the methylation continues as described in section 6.3. 1.5 mikrol
the resulting solution is injected into the chromatograph.
7. GAS CHROMATOGRAPHY
The column must yield a resolution "R" equal to, or better than, 1.5, where:
(d) ' (r2-r1)
R = 2 -------------
W1 + W2
where
R1 and r2 = retention times, two peaks (min.)
W1 and W2 = peak widths at half height (in millimetres),
d ' = the chart speed (mm/min).
As appropriate, showed the following chromatographic conditions:
Column: stainless steel.
Length: 1.7 m.
Diameter: 3 mm.
Carrier:
Chromosorb WAW
grit: 80 to 100 mesh.
Stationary phase: 10% OV 17.
Temperature:
column: 280 ° C,
Injector: 280 ° C,
detector: 280 ° C.
Carrier gas: nitrogen free oxygen.
Pressure: 2.3 bar.
Flow rate: 30 ml/min.
8. The CALCULATION of the
the proportionality Coefficient of hexachlorophene 8.1.
Will match the selected Standard and its ratio in the standard mixture.
If the
h = the hexachlorophene,
KH = its proportionality coefficient,
m ' h = its mass in the mixture (in g)
And ' h = its peak area,
s = the chosen standard,
m's = its mass in the mixture (in g)
A's = its peak area,
then:
m ' h's
kh = --- x ----
m's and ' h
5.1 the amount of hexachlorophene in the sample
If the
h = the hexachlorophene,
KH = its proportionality coefficient,
Ah = its peak area,
s = the chosen standard,
Ms = its mass in the mixture (in g)
As = its peak area,
M = weight of sample (g),
then the% (m/m) of hexachlorophene in the sample is
Ms x x x 100 Ah kh
-------------------
M-x Inc.
8. REPEATABILITY
For products containing about 0.1% (m/m) of hexachlorophene, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.005%.
20. The QUANTITATIVE DETERMINATION of TOSYLCHLORAMIDE SODIUM,
(CHLORAMINE-T) (INN)
1. scope and field of application
This method is described for the quantitative determination of tosylchloramide
sodium (chloramine-T) in cosmetic products by
a thin layer.
2. The DEFINITION of the
Chloramine-T content of the sample determined by this method is expressed in
percentage by mass (m/m).
3. The PRINCIPLE of the
Chloramine-T is quantitatively hydrolyzed to 4-toluenesulphonamide by boiling with
with hydrochloric acid.
The amount of the resulting 4-toluenesulphonamide chromatography on thin
layer provides fotodenzitometricky.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1 Tosylchloramide sodium (chloramine-T)
4.2. Standard solution of 4-toluenesulphonamide: 50 mg of 4-toluenesulphonamide in
100 ml of ethanol (4.5)
4.3. hydrochloric acid, 37% (m/m), d204 = 1.18 g/ml
4.4. diethyl ether
4.5. Ethanol, 96% (V/V)
4.6 mobile phase
4.6.1 the Butan-1-ol/ethanol (4.5)/water (40:4: 9; V/V/V), or
4.6.2 Chloroform/acetone (6:4, V/V)
4.7 the finished plates for thin-layer chromatography, silica gel 60, without
fluorescence indicator
4.8 the potassium permanganate
4.9. hydrochloric acid, 15% (m/m)
4.10 the detection reagent: 2-toluidine, 1% solution (m/V) in ethanol (4.5)
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2 the normal equipment for thin-layer chromatography
5.3 Fotodenzitometr
6. the procedure
6.1 the Hydrolysis
Flask of 50 ml round-bottomed weigh approximately 1 g of the sample (m).
Add 5 ml of water and 5 ml of hydrochloric acid (4.3) and one
an hour under a reflux condenser. Hot suspension immediately converts the water
the volumetric flask, 50 ml. Leave to cool and make up to the mark with
water. Centrifuge for 5 minutes at least 3000 Rev/min and the supernatant
filter.
6.2 Extraction
6.2.1.
With 30 ml of the filtrate and extract three times with 15 ml of diethyl ether
(4.4) if necessary, the ether phase dried and collected in 50 ml
a volumetric flask. Make up to volume with diethyl ether (4.4).
6.2.2.
Take 25 ml of the dried Ethereal extract and evaporate to dryness in a
a stream of nitrogen. Dissolve the residue in 1 ml of ethanol (4.5).
6.3. Thin-layer chromatography
6.3.1.
20 mikrol of the ethanolic solution (6.2.2) odparku is applied to the plate for the
thin-layer chromatography (4.7).
At the same time and in the same manner, apply 8, 12, 16 and 20 mikrol
standard solution of 4-toluenesulphonamide (4.2).
6.3.2.
Develop in the mobile phase (4.6.1 or 4.6.2) up to the brow reaches the
a distance of about 150 mm.
6.3.3.
After the complete evaporation of the mobile phase plate for 2 to 3 minutes to
the atmosphere par chlorine, incurred by pouring about 100 ml of acid
hydrochloric acid (4.9) to about 2 g of potassium permanganate (4.8)
a closed container. Excess chlorine is removed by heating the plate at 100th.
(C) for 5 minutes. Then board the visualizing agent (4.10).
6.4 Measurement
After approximately one hour with the purple spots by fotodenzitometrem
at 525 nm.
6.5 the Pronouncement of the calibration curves
The maximum peak height values specified for the four spots 4-
toluenesulphonamide spots against the corresponding quantities of 4-
toluenesulphonamide (i.e. 4, 6, 8 and 10 of 4-toluenesulphonamide mikrog on
the stain).
7. the NOTE
The method can be verified using the 0.1 or 0.2% solution (m/V) of chloramine-T
(4.1), to which are subjected to the same procedure as the sample (6).
8. The CALCULATION of the
Chloramine-T content of the sample, in percentage by mass,
calculated as follows
1.33 x and
% (m/m) tosylchloramide sodium =-----------
60 x m
where
1.33 = the 4-toluenesulphonamide by conversion factor/Chloramine-T,
a = quantity of 4-toluenesulphonamide in the sample, read
from the calibration curves (mikrog)
m = weight of sample (g).
9. REPEATABILITY
For products containing about 0.2% (m/m) of chloramine-T, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.03%.
21. The QUANTITATIVE DETERMINATION of TOTAL fluorine in dental creams
1. scope and field of application
This method is intended for the quantitative determination of total fluorine in
toothpastes. It is appropriate for the content not exceeding 0.25%.
2. The DEFINITION of the
Fluorine content of the sample determined by this method is expressed in
percentage by weight.
3. The PRINCIPLE of the
The determination is done by gas chromatography. Fluorine compounds of
fluorine-containing triethylfluorosilane (TEFS) direct response with
chlorotriethylsilane (TECS) in acidic environment and simultaneously
with xylene containing cyclohexane as internal standard.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Sodium fluoride, dried at 120 ° C to constant weight
4.2 Water, double distilled or of equivalent quality
4.3. hydrochloric acid, d204 = 1.19 g/ml
Cyclohexane (C6H12) 4.4
4.5 the Xylene
not showing on the chromatogram in chromatography under the same conditions
as the sample (6.1) no peaks before the peak of the solvents. In case of need
to clean a distillation (5.8).
4.6 Chlorotriethylsilane (TECS Merck or equivalent)
4.7 fluorine standard solutions
stock solution 4.7.1, 0,250 mg F-/ml.
Weigh accurately 138.1 mg of sodium fluoride (4.1) and dissolve in water
(4.2). Quantitatively transfer the solution into a 250 ml volumetric flask
(5.5). Dilute to the mark with water (4.2) and mix thoroughly.
4.7.2 the diluted stock solution,
0.050 mg F-/ml. In a 100 ml volumetric flask (5.5) with a pipette, converts 20
ml of the stock solution (4.7.1). Dilute to the mark with water and mix.
4.8. Internal standard solution
Mix 1 ml of cyclohexane (4.4) and 5 ml of xylene (4.5).
4.9 chlorotriethylsilane/internal standard
Into volumetric flasks 10 ml pipette (5.7) 0.6 ml of TECS (4.6) and
0.12 ml of internal standard solution (4.8). Dilute to the mark and xylene (4.5)
mix. Prepare fresh every day.
4.10 perchloric acid, 70% (m/V)
4.11 perchloric acid, 20% (m/V) in water (4.2)
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2. Gas Chromatograph fitted with a flame ionization detector
5.3 Vortex Mixer or equivalent
5.4 Bühler, type SMB1 or equivalent
5.5 volumetric flasks, 100 and 250 ml polypropylene
5.6
Centrifuge tube (glass) to 20 ml, with screw caps
coated with Teflon, Sovirel type 611-56 or equivalent. Cells and
closures to clean the extraction process in perchloric acid (4.11) in a few hours,
then rinse out the water five times (4.2), and finally dry at 100 ° C.
5.7 Pipettes with an adjustable volume of 50 to 200 mikrol, the peaks of the
plastic Disposable.
5.8 the distillation apparatus with a three-ball Schneider column or
equivalent Vigreux column column.
6. the procedure
6.1 analysis of sample
6.1.1.
Selects the still unopened tube of toothpaste, and cut all the
the content is removed. Transfer to a plastic container, mix thoroughly and
shall be kept under conditions in which there is no spoilage.
6.1.2.
Into the centrifuge tube (5.6) weigh accurately 150 mg of the sample (m),
Add 5 ml of water (4.2) and homogenize (5.3).
6.1.3
Add 1 ml of xylene (4.5).
6.1.4.
Drop by drop, add 5 ml of hydrochloric acid (4.3) and homogenise
(5.3).
6.1.5
In the centrifuge tube (5.6), add 0.5 ml of the solution and pipette
chlorotriethylsilane/internal standard solution (4.9).
6.1.6
Furnace with screw cap (3.5) and shake vigorously for 45
minutes on the shaker (5.4) set to 150 vibrations per minute.
6.1.7
Centrifuge for 10 minutes at that speed, in order to clear
the separation of the phases, uncap, the organic layer is separated and 3 mikrol
the organic phase into the gas chromatograph (5.2).
Note:
The elution of all the ingredients takes about 20 minutes.
6.1.8.
Repeat the injection, calculate the average peak area ratio (ATEFS/ACH), and
the corresponding content of fluorine (in milligrams (m1)) read from the calibration curve
(6.3).
6.1.9
The total fluorine content of the sample (in percentage by mass of fluorine)
calculated in the manner described in paragraph 7.
6.2. Chromatographic conditions
6.2.1 column: stainless steel.
Length: 1.8 m.
Diameter: 3 mm.
Gaschrom Q 80 to 100 mesh.
Stationary phase: silicon oil DC 200 or equivalent, 20%.
Then condition the column overnight at 100 ° C (at a flow rate of carrier
gas 25 ml nitrogen per minute), which is repeated every night. After each
the fourth or fifth injection recondition on
100 ° C for 30 minutes.
Temperature mode:
column: 70 ° C,
Injector: 150 ° C,
the detector. 250 ° C.
The carrier gas flow: 35 ml of nitrogen per minute.
6.3 calibration curve
6.3.1.
In a series of six centrifuge tubes (5.6), pipette 0, 1, 2, 3,
4 and 5 ml of the diluted fluoride standard solution (4.7.2). Each
close the tube with water (4.2) on a volume of 5 ml.
6.3.2.
Proceed as described under 6.1.3 to 6.1.6 inclusive.
6.3.3.
The gas chromatograph (5.2) inject 3 mikrol
the organic phase.
6.3.4.
Repeat the injection and calculate the average peak area ratio (ATEFS/ACH).
6.3.5.
Plot a calibration curve of weight of fluorine (in milligrams) in the
the standard solutions (6.3.1) and the peak area ratio (ATEFS/ACH) measured
referred to in section 6.3.4. By using regression analysis shall be the best spacing
the points of the curve line.
7. The CALCULATION of the
The concentration of the total fluorine in the sample (in percentage by weight
fluorine) (% (m/m) F) is given by
M1
% F =-x 100%
m
where
m = the test sample (6.1.2) (in mg)
M1 = the amount of F (in milligrams) read from the calibration graph (6.1.8).
8. REPEATABILITY
For products containing about 0.15% (m/m) the difference between the fluorine
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.012%.
22. The QUANTITATIVE DETERMINATION of ORGANIC COMPOUNDS of MERCURY
SCOPE AND FIELD OF APPLICATION
Furthermore, you can use the method described for the qualitative and quantitative
determination of organic derivatives of mercury used as a preservative
the ingredients in cosmetic products and make-up Remover for eyes. Method
can be used for thiomersal (INN), merthiolát and phenylmercuric salts.
AND QUALITATIVE DETERMINATION
1. The PRINCIPLE of the
Organic mercury compounds are converted to complex with 1, 5difenyl-
3thiokarbazonem. After extracting the dithizonátu carbon tetrachloride shall be
thin-layer chromatography on silica gel. Dithizonáty on
a chromatogram as orange spots.
2. USED CHEMICALS
All the reagents must be of analytical purity or higher
2.1 the sulphuric acid 25% (V/V)
2.2 l 5-diphenyl-3-thiocarbazone (dithizone): 0.8 mg in 100 ml
carbon tetrachloride (2.4)
2.3 Nitrogen
2.4. Carbon tetrachloride
2.5. Mobile phase: hexane/acetone, 90:10 (V/V)
2.6
Standard solution, 0.001% in water:
2-(ethylmercuriothio) benzoate, sodium
ethylrtuti chloride or methylmercury chloride,
fenylrtuti nitrate or acetate, fenylrtuti,
mercuric chloride or mercuric acetate.
2.7 Finished silica gel plates (e.g.. Merck 5721 or equivalent)
2.8. sodium chloride
3. apparatus and EQUIPMENT
3.1. Normal laboratory equipment
3.2. Usual TLC equipment
3.3 phase separation filter
4. the procedure
4.1 Extraction
4.1.1
l g of sample in a centrifuge tube, dilute 20 ml of distilled water. What
the most dispersed and heated in a water bath at 60 ° C add 4.
g of sodium chloride (2.8). Shake. Allow to cool.
4.1.2
Centrifuge for at least 20 minutes at 4500 rpm, to separate the greater
part of the solid from the liquid. Filter into a separating funnel and add
with 0.25 ml of sulphuric acid (2.1).
4.1.3.
Extract several times with 2 to 3 ml of dithizone solution (2.2) until the
the last organic phase remains green.
4.1.4
All of the organic phase sequentially through the filter for the separation
phase (3.3).
4.1.5.
Evaporate to dryness in a stream of nitrogen (2.3).
4.1.6
Dissolved in 0.5 ml of carbon tetrachloride (2.4). The solution immediately
used in the manner described in section 4.2.1.
4.2 separation and qualitative determination
4.2.1.
On the silica gel plate (2.7) is immediately spotted 50 mikrol a solution in
carbon tetrachloride, prepared in accordance with section 4.1.6. At the same time, 10 ml
standard solution (2.6) shall be subjected to the procedure described in section 4.1 and 50
mikrol solution is spotted on the same plate.
4.2.2.
The Board is placed in the mobile phase (2.5) and the mobile phase is off
to a height of 150 mm. Organic mercury compounds appear as Orange
spots, whose colour is stable, if immediately after the evaporation of
the solvent shall cover the glass plate.
You can get, for example, the following Rf values:
-------------------------------------------------------
Rf Color
-------------------------------------------------------
Thiomersal 0.33 Orange
Chloride ethylrtuti 0.29 Orange
Methylmercury chloride 0.29 Orange
Salt fenylrtuti 0.21 Orange
Rtuťnaté salt 0.10 Orange
Mercuric acetate 0.10 Orange
1.5-diphenyl-3-thiocarbazone 0.09 Pink
-------------------------------------------------------
(B). THE QUANTITATIVE DETERMINATION OF
1. The DEFINITION of the
The contents of organic mercury compounds determined by this method is expressed in
percentage by mass (m/m) of mercury in the sample.
2. The PRINCIPLE of the
Method is based on the measurement of the total content of mercury present. It is therefore
necessary first to make sure that it is not the presence of inorganic mercury, and
identify organic compounds of mercury present in the sample. After
mineralization is relaxed mercury provides flameless atomic absorption.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 concentrated nitric acid, d204 = 1.41 g/ml
3.2. Sulphuric acid, d204 = 1.84 g/ml
3.3 double distilled water
3.4. Potassium permanganate, 7% solution (m/V)
3.5 Hydroxylamoniumchlorid, 1.5% solution (m/V)
3.6. potassium Peroxodisíran, 5% solution (m/V)
3.7 the stannous chloride, 10% solution (m/V)
3.8 concentrated hydrochloric acid, d204 = 1.18 g/ml
2.4 glass wool impregnated with palladnatým, 1% (m/m)
4. apparatus and EQUIPMENT
4.1. Normal laboratory equipment
4.2. Apparatus for flameless atomic absorption mercury determination
spectrometry (cold vapour technique), including the necessary
glass Cookware. Optical distance of at least 100 mm in length.
5. the procedure
Retain all the usual security measures for micro-
analysis of mercury.
5.1 the decomposition
5.1.1.
Weigh accurately 150 mg of the sample (m). Add 10 ml of nitric acid
(3.1) and leave to act for three hours in an airtight container in the water
bath at 55 ° C, at regular intervals. At the same time
carry out a blank test on the reagents.
5.1.2.
When cool, add 10 ml of sulphuric acid (3.2) and once again
heat for 30 minutes in a water bath at 55 ° C.
5.1.3.
Insert the container in an ice bath and add carefully 20 ml of water (3.3).
5.1.4
In batches, add 2 ml of 7% potassium permanganate solution (3.4),
until the solution will be left. Returns the next 15 minutes in the water
bath at 55 ° C.
5.1.5.
Add 4 ml of the potassium peroxodisíranu solution (3.6). In a warmup to the
a water bath at 55 ° C, for 30 minutes.
5.1.6.
Allow to cool and transfer to a 100 ml volumetric flask.
Rinse the beaker with 5 ml of hydroxylammonium chloride (3.5) and then
four times with 10 ml of water (3.3). The solution should be completely decolorized. Make up
the mark with water (3.3).
5.2 quantitative determination
5.2.1.
10 ml of the test solution (5.1.6) is transferred to glass vials for
determination of mercury by cold vapour technique (4.2). Dilute with 100
ml of water (3.3) and then add 5 ml of sulphuric acid (3.2) and 5 ml
stannous chloride solution (2.3). Mix after each addition. Waits
30 seconds to all ions of mercury fell to metallic mercury, and
then subtract the resulting value (n).
5.2.2.
Glass wool impregnated with paladnatým chloride (3.9) between
the mercury reduction vessel and cell apparatus (4.2). The procedure referred to in point
5.2.1 shall be repeated and the resulting value shall be deducted. If the value is not
zero mineralization was incomplete and the analysis must be repeated.
7. The CALCULATION of the
If the
m = mass of the test sample (mg)
n = the quantity of mercury that read on the machine (in mikrog)
The quantity of mercury, expressed in percentage by weight of mercury is calculated from the
formula:
m
% of mercury =--
n
7. NOTES
7.1 to improve mineralization it might be necessary to start by diluting the sample.
7.2 if there is suspicion that the mercury absorbed on the substrate, it should
perform a quantitative determination by the method of standard additions.
8. REPEATABILITY
For products containing about 0.007% (m/m) of mercury, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.00035%.
23. The QUANTITATIVE DETERMINATION of ALKALI SULPHIDES and METAL SULFIDES
ALKALINE EARTH METALS
1. scope and field of application
This method is described in the quantitative determination of sulfides present in the
cosmetic products. The presence of thiols or other reduction
reagents (including sulphites) does not interfere.
2. The DEFINITION of the
The concentration of sulphides determined by this method is expressed as percentage by weight
percentage of sulphur.
3. The PRINCIPLE of the
In an acidified hydrogen sulfide environment will take a current of nitrogen and then
precipitated in the form of cadmium sulphide. The liquid, wash with and
be determined iodometrically.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher
4.1. Concentrated hydrochloric acid, d204 = 1.19 g/ml
4.2. Sodium Thiosulphate, 0.1 mol/l standard solution
4.3. Iodine, 0.05 mol/l standard solution
4.4. Sodium sulfide
4.5 cadmium Acetate
4.6 Concentrated ammonia, d204 = 0.90 g/ml
4.7 Ammonia cadmium di(acetate) solution:
10 g of cadmium di(acetate) solution (4.5), dissolve in about 50 ml of water.
Add ammonia (4.6) until the precipitate is dissolved (i.e., approximately
20 ml). Make up to volume with water to 100 ml.
4.8. Nitrogen
4.9 M ammonia solution
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2 the ground glass Neck flask 100 ml round-bottom
5.3 Two Erlenmeyer flask with ground on 150 ml, with the tube for
supply and discharge gas.
5.4 One funnel with a long stem
6. the procedure
6.1 Release of sulfides
6.1.1.
Apply an unopened package resource. In the round-bottomed flask
(5.2) accurately weigh the quantity of product (m) (expressed in g)
corresponds to no more than 30 mg of sulphide ions. Add 60 ml of water and 2 drops
anti-foaming agents.
6.1.2.
In both Erlenmeyerových flasks (5.3), transfer 50 ml of the solution (4.7).
6.1.3
On tříhrdlou the flask (5.2) connects the separating funnel, tube inlet
and exhaust gas. Tube for exhaust gas connects via hoses of PVC
the conical flasks (5.3) in a row.
Note:
The equipment on the release of sulfides must pass the following test
leakage: under the same test conditions, which is to be
examined, 10 ml of a sulphide solution replaces the (prepared in accordance with section 4.4),
containing "X mg" of sulphide (iodometrically determined). Let the "Y" is the
the number of milligrams of sulphide found at the end of this operation. The difference between the
the amount of "X" and "Y" must not exceed 3%.
6.1.4.
To remove air, round-bottomed flask (5.2) 15 minutes
Browse the nitrogen (4.8) at 2 bubbles per second.
6.1.5
Round-bottomed flask is heated to 85 +/-5 ° C.
6.1.6
The nitrogen (4.8) stream will stop and drop by drop, add 40 ml of acid
hydrochloric acid (4.1).
6.1.7
The nitrogen (4.8) stream is reset after you add the acid, nearly all the leaves
the minimum quantity of the liquid, to prevent the leakage of hydrogen sulphide.
6.1.8.
After 30 minutes, the heating is broken. Flask (5.2) to allow at least an hour and
a half to cool for standing the nitrogen (4.8) stream.
6.2. Titration
6.2.1.
Cadmium sulphide precipitate is filtered through the funnel with a long
stem (5.4).
6.2.2.
Conical flask (5.3) first with the ammonia solution
(3.0) and the liquid is poured on the filter. Then rinse with distilled
water and the water used to wash the precipitate in the filter on.
6.2.3.
The washing of the precipitate is finished by washing the other 100 ml of water.
6.2.4
Filter paper is inserted into the first conical flask that contained the
the clot. Add 25 ml (n1) of the iodine solution (4.3), approximately 20 ml
hydrochloric acid (4.1) and 50 ml of distilled water.
6.2.5
Excess iodine lays down the Sodium Thiosulphate solution (n2) (4.2).
7. The CALCULATION of the
The content of sulfides in the sample, in percentage by mass of sulphur
calculated according to the following formula:
32 (n1 n2 x 1-x 2)
% síry = ------------------------
20 m
where:
N1 = the number of standard iodine solution (4.3)
(in millilitres),
x 1 = the Molarity of this solution,
N2 = the number of standard solution of Sodium Thiosulphate
(4.2) (in millilitres),
x 2 = the Molarity of this solution,
m = mass of the test sample (g).
8. REPEATABILITY
For products containing about 2% (m/m) the difference between the results of sulfides
two parallel on the same sample should not exceed the determination of absolute
the value of 0.2%.
24. The qualitative and QUANTITATIVE DETERMINATION of 2.3-DIHYDROXYPROPYL-4-
AMINOBENZOATE
AND QUALITATIVE DETERMINATION
1. scope and field of application
This method is used for qualitative determination of 2.3-dihydroxypropyl-
4-aminobenzoate (glycerol1 (4aminobenzoátu)). Also serves to
qualitative determination of ethyl 4-aminobenzoate (benzocaine INN), which
may be present as an impurity.
2. The PRINCIPLE of the
Qualitative determination is done by thin layer chromatography with
the use of silica gel and fluorescent indicator and free
a primary amine group by formation of a diazo dye on the plate.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 the solvent mixture: cyclohexane/propan2ol/stabilized dichloromethane
48/64/9 (V/V/V).
3.2. mobile phase: light petroleum (4060)/benzen/aceton/roztok hydroxide
(at least 25% ammonia): 35/35/35/1 (V/V/V/V).
3.3
Detection reagent:
and) sodium nitrite: 1 g in 100 ml of 1 m HCl (prepared just before
use),
(b)) 2naftol: 0.2 g in 100 ml of 1 m potassium hydroxide.
3.4 standard solutions:
2.3-dihydroxypropyl-4-aminobenzoate: 0.05 g in 100 ml of mixed
the solvent 3.1;
4-aminobenzoate: 0.05 g in 100 ml of mixed solvent 3.1.
3.5 the silica gel 60 F254 plates, plates of a thickness of 0.25 mm thick, 200 mm x 200
mm.
4. apparatus and EQUIPMENT
4.1. Normal apparatus for thin-layer chromatography
4.2. ultrasonic bath
Millipore filter FH 0.5 4.3 mikrom or equivalent
5. the procedure
5.1. Preparation of the sample
In a volumetric flask with 10 ml weigh 1.5 g of the product
to be analyzed. Make up to the mark with the solvent 3.1. Stopper and 1
leave at room temperature for an hour in an ultrasonic vibrator (4.2).
Filter through a Millipore filter (4.3) and use the filtrate for the
chromatography.
5.2. Thin-layer chromatography
On the plate (3.5) apply 10 mikrol of the sample solution (5.1) and each of the
the standard solutions (2.1). The chromatogram to a height of 150 mm in
the Chamber previously saturated with mobile phase 3.2. Allow the plate to dry at
temperature around.
5.3 Detection
5.3.1 the plate under UV light 254 nm.
5.3.2 the Completely dried plate with the solution 3.3 and detection).
Allow the plate to dry for 1 minute at room temperature and immediately
spray the visualizing the solution 3.3 (b)). Dry in an oven at 60 ° C.
The spots will appear orange. 2.3-dihydroxypropyl-4-aminobenzoate:
RF about 0.07, 4-aminobenzoate: Rf 0.55.
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
This method is used for the quantitative determination of 2.3-dihydroxypropyl-
4-aminobenzoate also establishes 4-aminobenzoate. Cannot be determined
more than 5% (m/m) of 2.3-dihydroxypropyl-4-aminobenzoate and introduce it more than 1%
(m/m) ethyl 4-aminobenzoate.
2. The DEFINITION of the
The contents of the 2.3-dihydroxypropyl-4-aminobenzoate and ethyl 4-aminobenzoate contents
set by this method is expressed in percentage (% m/m) weight
resource.
3. The PRINCIPLE of the
A resource that has to be analyzed is suspended in methanol and after
suitable treatment of the sample is determined by high-performance liquid
chromatography (HPLC).
4. CHEMICALS USED
All the reagents must be of analytical purity or higher and must be
suitable for HPLC.
4.1 Methanol
4.2. Potassium dihydrogen phosphate (KH2PO4)
4.3 zinc acetate (Zn (CH3COO) 2.2H2O)
4.4. acetic acid (d204 = 1.05)
4.5. Potassium hexacyanoferrate (K4 (Fe (CN) 6) .3H2O)
4.6 Ethyl4hydroxybenzoát
2.3-dihydroxypropyl 2.9-4-aminobenzoate
4.8 4-Aminobenzoate
4.9. phosphate buffer (0.02 m):
dissolve 2.72 g of potassium dihydrogenorthophosphate (4.2) in a 1 litre
water.
4.10. mobile phase: phosphate buffer solution (4.9)/methanol (4.1) 61/39
(IN/IN)
The composition of the mobile phase may be changed in order to achieve a factor of
resolution R > = 1.5.
d ' R2-d ' R1
R = 2 ----------------
W1 + W2
where
R1 and R2 = retention times, in minutes, of the peaks.
W1 and W2 = peak widths at half height, in millimetres,
d ' = the chart speed in mm/min.
4.11 2.3-dihydroxypropyl stock solution-4-aminobenzoate:
Weigh 40 mg of 2.3-dihydroxypropyl-4-aminobenzoate, accurately and
transfer to a 100 ml volumetric flask dissolve in 40 ml of methanol
(4.1) make up to the buffer solution (4.9) and mix.
4.12. stock solution of ethyl 4-aminobenzoate:
Weigh accurately about 40 mg of ethyl 4-aminobenzoate and transferred into a graduated
flasks, 100 ml. Dissolve in 40 ml of methanol (4.1). Make up
buffer solution (4.9) and mix.
4.13. Internal standard solution:
Weigh accurately about 50 mg of ethyl 4-hydroxybenzoate, and transfer to
volumetric flasks, 100 ml. Dissolve in 40 ml of methanol (4.1). Make up
with the buffer solution (4.9) and mix.
4.14. Standard solutions:
Prepare four standard solutions by dissolving substances in the
100 ml of mobile phase (4.10) according to the following table:
|----------------------------------------------------------------------------------------|
| 2.3-dihydroxypropyl standard |-4-and 4-aminobenzoate | ethyl4hydroxybenzoát |
| | minobenzoát solution | | |
| |----------------------------------------------------------------------------|
| | (mikrog/ml) ml (4.11) | (mikrog/ml) ml (4.12) | (mikrog/ml) ml (4.13) |
| | (*) | | (*) | | (*) | |
|----------------------------------------------------------------------------------------|
| I |8 |2 |8 |2 |50 |10 |
| | 16 | 4 | 12 | 3 | 50 | 10 |
| (III) | 24 | 6 | 16 | 4 | 50 | 10 |
| IV | 40 | 10 | 20 | 5 | 50 | 10 |
|----------------------------------------------------------------------------------------|
| (*) These values are indicative and correspond to the exact masses in 4.11, 4.12 and 4.13. |
| Note: these solutions can be prepared in a different way. |
-----------------------------------------------------------------------------------------|
|
4.15. carrez solution I:
Dissolve 26.5 g hexacyanoferrate (4.5) in water and
make up to 250 ml.
4.16. carrez solution II:
Dissolve 54.9 g of zinc acetate (4.3) and 7.5 ml of acetic acid
(4.4) in water and make up to 250 ml.
Merck Lichrosorb RP18 4.17, or equivalent, with an average size of
5 mikrom
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2 high-performance chromatography equipment with a UV detector with
adjustable wavelength and the Chamber with the thermostat set at 45th.
(C)
5.3 stainless-steel Column: length: 250 mm; internal diameter: 4.6 mm;
Lichrosorb RP-18 filling (4.17)
5.4. ultrasonic bath
6. the procedure
6.1. Preparation of the sample
6.1.1.
The 100 ml beaker weigh 1 g of the sample and add 10 ml of
methanol (4.1).
6.1.2.
The beaker is placed on the 20 minutes in the ultrasonic bath (5.4), in order to
created the suspension. Thus the suspension quantitatively
a maximum of 75 ml of mobile phase (4.10) into a volumetric flask of 100 ml.
Gradually add 1 ml of carrez solution I (4.15) and 1 ml of carrez
reagent II (4.16) and mix thoroughly after each addition. Make up Elution
solution (4.10) with, again, mix thoroughly and filter through
pleated filter paper.
6.1.3
Into a 50 ml volumetric flask, pipette 3.0 ml of the filtrate obtained converts
According to paragraph 6.1.2 and 5.0 ml of internal standard solution (4.13). Make up
the mobile phase and mix. This solution is used
for the implementation of chromatographic analysis described in point 6.2.
6.2 the Chromatography
6.2.1.
The flow rate of the mobile phase (4.10) to 1.2 ml/min and set the column temperature
sets the 45 ° C.
6.2.2.
The detector (5.2) to 274 nm on.
6.2.3.
Into the chromatograph with a microsyringe, inject at least twice by 20 mikrol
solution (6.1.3) and measure the peak areas.
6.3 calibration curve
6.3.1.
Inject 20 mikrol each of the standard solutions (4.14) and measure the
peak areas.
6.3.2.
For each concentration calculate the ratio of the areas of the 2.3-
dihydroxypropyl-4-aminobenzoate, to the areas of the peaks of the internal standard.
This ratio is plotted on the x axis and the y axis is plotted on the ratio
the corresponding weight.
6.3.3.
Proceed in the same way in the case of ethyl 4-hydroxybenzoate.
7. The CALCULATION of the
7.1
From the calibration curve obtained in point 6.3 of the ratios of the masses of
(RP1, RP2) corresponding to the ratios between the areas of the peaks calculated in point
6.2.3 where
RP1 = mass of 2.3-dihydroxypropyl-4-aminobenzoate/hmotnost ethyl-
4hydroxybenzoátu,
Rp2 = mass of ethyl-4aminobenzoátu/weight of ethyl 4-hydroxybenzoate,
7.2.
The ratios thus obtained mass content of 2.3-
dihydroxypropyl-4-aminobenzoate and ethyl 4-aminobenzoate contents in
percentage by mass (% m/m), using the formula:
q
RP% (m/m) of 2.3-dihydroxypropyl-4-aminobenzoate = RP2 ×----
6 p
q
RP% (m/m) ethyl 4-aminobenzoate = RP2 ×----
6 p
q = quantity of ethyl 4-hydroxybenzoate (internal standard)
weighed in point 4.12 mg,
p = quantity of sample, weighed in 6.1.1 in the (g).
8. REPEATABILITY
8.1.
For products containing about 5% (m/m) of 2.3-dihydroxypropyl-4-
aminobenzoate, the difference between the results of two parallel determinations on
the same sample should not exceed an absolute value of 0.25%.
8.2.
For products that contain about 1% (m/m) ethyl 4-aminobenzoate, the difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.10%.
9. NOTES
9.1.
Before analysis it is necessary to check whether the sample contains
substances whose peaks could interfere with internal standard (ethyl-
4aminobenzoát).
9.2
To check that there is no further interference, repeat the determination,
While the proportion of methanol in the mobile phase changes relatively by 10%.
25. The QUANTITATIVE DETERMINATION of CHLOROBUTANOL
1 scope and field of application
This method is suitable for the quantitative determination of chlorobutanol (INN)
up to a maximum concentration of 0.5% (m/m) in all cosmetic
resources with the exception of aerosols.
2 definitions
The content of chlorobutanol determined by this method is expressed in percentage
weight (% m/m) of the resource.
3 PRINCIPLE
After the appropriate processing of the resource that is to be analyzed the determination
performs by gas chromatography with 2.2, 2trichlorethanolem as internal
standard.
4. USED CHEMICALS:
All the reagents must be of analytical purity or higher
4.1 Chlorobutanol (1,1, 1trichlor2methylpropan2ol)
4.2.2.2, 2trichlorethanol
4.3 the absolute ethyl alcohol
4.4. standard solution of chlorobutanol: 0.025 g in 100 ml ethanol (4.3)
(m/V)
4.5 2.2 standard solution, 2trichlorethanolu: 4 mg in 100 ml of ethanol
(4.3) (m/V)
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2. Gas Chromatograph with electron capture detection with a 63Ni
6. the procedure
6.1. Preparation of the sample
In a 100 ml volumetric flask, weigh from 0.1 to 0.3 g of the sample (p
grams). Dissolve in ethanol (4.3), add 1 ml of the solution
internal standard solution (4.5) and make up to the mark with ethanol.
6.2. Chromatographic conditions
6.2.1.
Operating conditions must be a distinguishing factor R > = 1.5.
(d) ' (R2-R1)
R = 2 -------------
W1 + W2
where
R1 and R2 = retention times, in minutes, of the peaks.
W1 and W2 = peak widths at half height, in millimetres,
d ' = the chart speed in mm/min.
6.2.2.
The resolution shall provide, for example, the following operating conditions:
------------------------------------------------------
Column I II
------------------------------------------------------
Material Glass stainless steel
------------------------------------------------------
Length 1.8 m 3 m
------------------------------------------------------
Diameter 3 mm 3 mm
------------------------------------------------------
The stationary phase 10% Carbowax 20 M 5% OV 17 on
TPA on Chromosorb WAW Gaschromu
Q 80-100 mesh DMCS 80-100
mesh
------------------------------------------------------
Conditioning 2 to 3 days when
190 ° C
------------------------------------------------------
Temperature:
-Injector 200 ° C 150 ° C
-column 150 ° C 100 ° C
-detector 200 ° C 150 ° C
------------------------------------------------------
Carrier gas Nitrogen Argon/methane
(95/5 V/V)
------------------------------------------------------
Flowrate 35 ml/min 35 ml/min
------------------------------------------------------
6.3 calibration curve
Into five 100 ml volumetric flasks, add 1 ml of the standard solution
(4.5) and 0.2, 0.3, 0.4, 0.5 0.6 ml of solution 4.4 or the volume make up
mark with ethanol (4.3) and mix. Inject into the chromatograph after 1
mikrol each of these solutions for the operating conditions described in point
6.2.2 and the calibration curve by plotting the ratio of the masses
chlorobutanol and 2.2, 2trichlorethanolu on the x-axis and the ratio of the corresponding
peak areas on the y axis.
6.4.
Inject 1 mikrol solution obtained in 6.1 and proceed
According to the conditions described in 6.2.2.
7. The CALCULATION of the
7.1
From the calibration curve (6.3) the value "and" expressed in mikrog
chlorobutanol, in the solution 6.1.
7.2.
The content of chlorobutanol in the sample is calculated according to the formula
and x 102 and
% chlorobutanol =----------------
p x p x 104 106
8. REPEATABILITY
For products containing about 0.5% (m/m) of chlorobutanol, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.10%.
Note:
If the result is equal to or greater than the maximum allowed
concentration, it is necessary to check whether there is interference.
26. The qualitative and quantitative DETERMINATION of QUININE
AND QUALITATIVE DETERMINATION
1. scope and field of application
This method is intended for the qualitative determination of the presence of quinine in
shampoos and hair lotions.
2. The PRINCIPLE of the
Qualitative determination is done by thin layer chromatography
silica gel. Is the blue fluorescence of quinine in acidic environment
at 360 nm.
For further confirmation, you can delete the vapours of bromine and fluorescence of steam
ammonia vapours will cause a yellow fluorescence.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 the silica gel plates, without fluorescence indicator, of thickness
layer 0.25 mm, 200 mm x 200 mm
3.2. mobile phase: toluene/diethyl ether/dichloromethane/diethylamine
20/20/20/8 (V/V/V/V)
3.3 Methanol
3.4 sulphuric acid (96%; d204 = 1.84)
3.5. diethyl ether
3.6 Detection reagent:
to 95 ml of diethyl ether (3.5) in a cooled container carefully add 5 ml of
sulphuric acid (3.4).
3.7 Brom
3.8 aminiaku aqueous solution (28%; d204 = 0.90)
2.4 Quinine, anhydrous
3.10. Standard solution:
volumetric flask, weigh accurately about 100.0 mg of anhydrous quinine (3.9)
and dissolve in 100 ml of methanol (3.3).
4. apparatus and EQUIPMENT
4.1 Normal equipment for thin-layer chromatography
4.2. ultrasonic bath
Millipore filter FH 0.5 4.3 mikrom or equivalent and appropriate filter
device
5. the procedure
5.1. Preparation of the sample
In a 100 ml volumetric flask, weigh accurately a quantity of the sample
contains about 100 mg of quinine, dissolve and make up to the mark with methanol
(3.3).
Stopper and leave for one hour at room temperature in
an ultrasonic vibrator (4.2). Filter (4.3) and use the filtrate
for chromatography.
5.2. Thin-layer chromatography
On the plate (3.1) 1.0 mikrol standard solution (3.10) and 1.0
mikrol of the sample solution (5.1). The chromatogram to a height of 150 mm in
Chamber in advance of saturated with solvent (3.2).
5.3 Detection
5.3.1.
The plate is dried at room temperature.
5.3.2.
Spray with reagent 3.6.
5.3.3.
Allow the plate to dry for one hour at room temperature.
5.3.4.
Observe in the UV lamp light set to a wavelength of 360 nm.
Quinine is reflected as intensely fluorescent blue spot.
As an example, are listed in the following table the values of the Rf of the main
alkaloids related to developing with solvent 3.2:
---------------------------------
Alkaloid Rf
---------------------------------
Quinine 0.20
Quinidine 0.29
Cinchonin 0.33
Cinchonidin 0.27
Hydrochinidin 0.17
---------------------------------
5.3.5
For further confirmation of the presence of quinine, the Board will issue about one
an hour to bromine vapour (3.7). The fluorescence disappears. They rode the same motherboard
exposed to ammonia vapour (3.8), the spots reappear in brown colour and
under the UV lamp (360 nm) is a yellowish fluorescence.
Detection limit: 0.1 mikrog quinine.
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
This method is described for the quantitative determination of quinine. Method can be
used to determine the maximum permitted concentration of 0.5%
(m/m) in shampoos and 0.2% in hair lotions.
2. The DEFINITION of the
The quinine content determined by this method is expressed in percentage by weight
(% m/m) of the resource.
3. The PRINCIPLE of the
After the appropriate processing of the resource that is to be analyzed the determination
performs high-performance liquid chromatography (HPLC).
4. CHEMICALS USED
All the reagents must be of analytical purity or higher and must be
suitable for HPLC.
4.1 Acetonitrile
4.2. Potassium dihydrogen phosphate (KH2PO4)
4.3 orthophosphoric acid (85%; d204 = 1.7)
4.4 Tetramethylamoniumbromid
4.5 Quinine, anhydrous
4.6 Methanol
4.7. orthophosphoric acid acid solution (0,1 m):
Weigh 11.53 g of orthophosphoric acid (4.3) and dissolve in
in a 1000 ml volumetric flask of water.
4.8. Solution of potassium dihydrogen phosphate (0,1 m):
Weigh 13.6 g of potassium dihydrogenorthophosphate (4.2) and dissolve
in a graduated flask 1000 ml of water.
tetramethylammonium bromide Solution: 4.9
Dissolve 15.40 g tetramethylammonium bromide (4.4) in a volumetric flask
1000 ml of water.
4.10. Mobile phase:
Orthophosphoric acid (4.7)/roztok dihydrogen phosphate
potassium (3.0)/roztok tetramethylamonium bromide
(3.0)/voda/acetonitril (4.1) 10/50/100/340/90 (V/V/V/V/V).
Composition of the mobile phase may be changed in order to achieve
resolution R > = 1.5.
(d) ' (R2-R1)
R = 2 -------------
W1 + W2
where
R1 and R2 = retention times, in minutes, of the peaks.
W1 and W2 = peak widths at half height, in millimetres,
d ' = the chart speed in mm/min.
4.11 the silica gel oktadecylsilylovaný, 10 mikrom
4.12 standard solutions:
Into 100 ml volumetric flasks weigh about 5.0; 10.0; 15.0 and 20.0 mg
respectively of quinine anhydrous (4.5), make up to the mark with methanol (4.6) and
shake contents until the quinine dissolves. Solutions filter over the
filter 0.5 mikrom.
5. apparatus and EQUIPMENT
5.1. Normal laboratory equipment
5.2. ultrasonic bath
5.3. Device for high performance liquid chromatography detector
with a variable wavelength
3.4 column: length 250 mm, internal diameter 4.6 mm, filling: silica gel
(4.11)
Millipore filter FH 0.5 3.4 mikrom or equivalent with suitable
filter device
6. the procedure
6.1. Preparation of the sample
In a 100 ml volumetric flask, weigh accurately a quantity of the resource, which
contains about 10.0 mg of anhydrous quinine, add 20 ml of methanol (4.6)
and the bottle is immersed for 20 minutes in an ultrasonic bath (5.2). The volume of the
make up to the mark with methanol (4.6), mix and an aliquot is
filter (5.5).
6.2. Chromatographic conditions
Flowrate: 1.0 ml/min.
Detector wavelength (5.3): 332 nm.
Injection volume: 10 mikrol of the filtered solution (6.1).
Measured variable: peak area.
6.3 calibration curve
At least three times to inject 10 mikrol each standard solution
(4.12), measure the area of the peaks, and calculate the mean value for each
concentration.
Plot the calibration curve and verify that it forms a straight line.
7. The CALCULATION of the
7.1
From the calibration curve (6.3) the quantity of anhydrous quinine in the
mikrog, contained in 7.1.from (6.2).
7.2.
The concentration of anhydrous quinine in the sample, expressed in weight
percentages (% m/m) is calculated from the formula:
(B)
% (m/m) of anhydrous quinine =--
And
where
(B) = the quantity of anhydrous quinine in mikrog found
in the 10 microlitres of the filtered solution (6.1),
A = weight of sample in g (6.1).
8. REPEATABILITY
For products containing about 0.5% (m/m) of anhydrous quinine difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.02%.
For products containing about 0.2% (m/m) of anhydrous quinine difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.01%.
27. The qualitative and quantitative DETERMINATION of INORGANIC SULPHITES and
HYDROGEN SULPHITES
SCOPE AND FIELD OF APPLICATION
This method describes the qualitative and quantitative determination of
inorganic sulphites and hydrogen sulphites in cosmetic
resource. The method is applicable only for resources that contain
aqueous or alcoholic phase and for concentrations of sulphur dioxide
not more than 0.2%.
AND QUALITATIVE DETERMINATION
1. The PRINCIPLE of the
The sample is heated with hydrochloric acid, and sulphur dioxide liberated
identifies its smell or indicator paper.
2. USED CHEMICALS
All the reagents must be of analytical purity or higher
2.1. hydrochloric acid 4 mol/l
2.2 with potassium iodate Starch paper or other appropriate
alternative
3. apparatus and EQUIPMENT
3.1. Normal laboratory equipment
3.2 25 ml flask with reflux condenser
4. the procedure
4.1
In the flask (3.2) with about 2.5 g of the sample and 10 ml of acid
(2.1).
4.2.
Mix and boil.
4.3.
Sulphur proves its smell or indicator
test.
(B). THE QUANTITATIVE DETERMINATION OF
1. The DEFINITION of the
The content of sulphite or hydrogen sulphite by mass in the sample, as determined by the
the method is expressed in percentage by mass of sulphur dioxide.
2. The PRINCIPLE of the
After acidification of the sample, sulphur dioxide liberated is accumulated in the peroxide solution
hydrogen. Resulting from the sulphuric acid to titrate the hydroxide
sodium.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Hydrogen peroxide 0.2% (m/V).
The solution is prepared fresh daily.
3.2 orthophosphoric acid (d204 = 1.75)
3.3 Methanol
3.4. Standard solution of sodium hydroxide 0, 01 m
3.5 Nitrogen
3.6 indicator:
mixture 1:1 (V/V) of methyl red (0.03% m/V in ethanol) and methylene
Blue (0.05% m/V) in ethanol. Filter the solution.
4. apparatus and EQUIPMENT
4.1. Normal laboratory equipment
4.2 the distillation apparatus (see fig. Apparatus for distillation of carbon
sulphur dioxide by Tanner)
5. the procedure
5.1
To the distillation flask A (see fig. Apparatus for distillation of sulphur dioxide
According to Tanner) weigh about 2.5 g of the sample accurately.
5.2.
Add 60 ml of water and 50 ml of methanol (3.3) and mix.
5.3.
To the distillation master D (see fig. Apparatus for distillation of carbon
sulphur dioxide by Tanner) place 10 ml of hydrogen peroxide solution
(3.1), 60 ml of water and add a few drops of indicator (3.6) and further
a few drops of sodium hydroxide (3.4) until the indicator green.
5.4.
The procedure referred to in 5.3 is repeated with the wash bottle E (see figure. The unit on the
the distillation of sulphur dioxide by Tanner).
5.5
Apparatus and nitrogen (3.5) flow to about 60 bubbles
per minute.
5.6
To the distillation flask and funnel of 15 ml of acid
orthophosphoric acid (3.2).
5.7
Heat to boiling and then simmer gently for 30 minutes.
3.6
The distillation master (D) is disconnected. Rinse the tube and
titration with sodium hydroxide solution (3.4) until the Green coloring
indicator (3.6).
6. The CALCULATION of the
The content of sulphite or hydrogen sulphite by mass in the sample by weight
the percentage is calculated from the formula:
3.2 MV
% (m/m) of sulphur dioxide =-------------
m
where
M = molar concentration of sodium hydroxide solution (3.4),
V = volume of sodium hydroxide solution (3.4) used in the
titration (5.8), in millilitres,
m = mass of sample (5.1) in grams.
7. REPEATABILITY
For products containing about 0.2% (m/m) of sulphur dioxide the difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.006%.
Apparatus for distillation of sulphur dioxide by Tanner
All dimensions in mm
Spherical condenser with 10 balls
28. The qualitative and quantitative DETERMINATION of CHLORATES of the ALKALI METALS
SCOPE AND FIELD OF APPLICATION
This method describes the identification and determination of chlorates in the dental
toothpaste and other cosmetic products.
AND QUALITATIVE DETERMINATION
1. The PRINCIPLE of the
Chlorates are separated from other halates by thin and iodates
layer and identify with the oxidation of iodide to form iodine.
2. USED CHEMICALS
All the reagents must be of analytical purity or higher
2.1 standard solutions: aqueous solutions of potassium chlorate, bromate and
potassium iodate 0.2% m/V, freshly prepared
2.2. mobile phase: 28% of the ammonia solution (m/V) acetone/butanol (60/130/30
V/V/V)
2.3 potassium iodide, aqueous solution of 5% m/V
2.4 the starch solution 1 to 5% m/V
2.5. Hydrochloric acid, 1 mol/l
2.6 Ready for cellulose thin-layer chromatography
(thickness of layer 0.25 mm)
3. apparatus and EQUIPMENT
Normal equipment for thin-layer chromatography
4. the procedure
4.1
About 1 g of the sample with water, filter, and dilute to about 25
ml.
4.2.
On the plate (2.6) 2 mikrol solution (4.1) and 2 mikrol each of
the three reference solutions (2.1).
4.3.
Place the plate in a tank and develop the chromatogram
solvent (2.2) by ascending chromatography about three-quarters of the length of the
the plate (2.6).
4.4.
The plate from the tank and allow the solvent to evaporate. (Note:
It may take up to 2 hours.)
4.5.
The plate with potassium iodide (2.3) and allow about 5
minutes to dry.
4.6.
Spray the plate with starch solution (2.4) and allow to dry for about 5 minutes.
4.7 spray the plate with hydrochloric acid (2.5).
5. EVALUATION of the
Chlorates are present, it will appear after half an hour (or blue
Brown) spot with an Rf value approximately 0.7 to 0.8.
---------------------------------------
RF
---------------------------------------
Iodate 0 to 0.2
Bromate 0.5 to 0.6
Chlorate 0.7 to 0.8
---------------------------------------
It should be noted that bromates and iodates give immediate reaction.
Care must be taken to not switched spots bromate and chlorate.
(B). THE QUANTITATIVE DETERMINATION OF
1. The DEFINITION of the
The content of chlorate of the sample determined by this method is expressed in
percentage by mass of chlorate.
2. The PRINCIPLE of the
Chlorate is reduced by zinc powder in an acid medium. The resulting
chloride is determined by potentiometric titration with a solution of nitrate
the silver. A similar determination before reduction reveals the possible presence of
bromates and iodates.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1 acetic acid, 80% (m/m)
3.2 the powdered zinc
3.3 Standard silver nitrate solution 0.1 mol/l
4. apparatus and EQUIPMENT
4.1. Normal laboratory equipment
4.2. Potentiometer with silver indicating electrode
5. the procedure
5.1. Preparation of the sample
In the centrifuge tube, weigh about 2 g of the sample (m grams).
Add about 15 ml acetic acid (3.1) and mix thoroughly. After 30
minutes of standing for 15 minutes at 2000 centrifuge RPM Supernatant
volumetric flasks, 50 ml. repeat Centrifugation
twice after adding 15 ml acetic acid (3.1) to the rest of the.
Supernatanty are merged in a single flask. Make up to the mark with
acetic acid (3.1).
3.2 Reduction of chlorate
To 20 ml of solution 5.1 Add 0.6 g of zinc powder (3.2). The content in the
the boiling flask under reflux must be brought to a boil. After 30 minutes of boiling
cool and filter. Rinse the flask with water, and wash the filter.
The filtrate and the washing water.
5.3. Determination of chlorides
20 ml solution 5.2 with silver nitrate solution (3.3)
potentiometric indication (4.2). In the same way, the solution
silver nitrate (3.3) Titrate 20 millilitres of solution 5.1.
Note:
Obsahujeli means of bromine or iodine derivatives which can release
When the bromides or iodides, the titration curve have several
points of inflection. In this case, the volume of titrant
corresponding to chloride given the difference between the last and the penultimate
Inflexion point.
6. The CALCULATION of the
The content of chlorate of the sample (% m/m) is calculated from the formula:
20.9 (V-V ') M
The content of chlorate (ClO3-)% m/m =--------------------
m
where
V = volume of the silver nitrate solution (3.3) in millilitres,
consumed in the titration solution 5.2,
In ' = the volume of silver nitrate solution (3.3) in millilitres,
used to titrate 20 ml of solution 5.1,
M = molality of silver nitrate standard solution (3.3),
m = weight of sample in g.
7. REPEATABILITY
For products containing about 3 to 5% (m/m) the difference between chlorate
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.07%.
29. The qualitative and quantitative DETERMINATION of SODIUM IODATE
SCOPE AND FIELD OF APPLICATION
This method describes the procedure for qualitative and quantitative determination of
sodium iodate in cosmetic products that after use
Rinse immediately.
AND QUALITATIVE DETERMINATION
1. The ESSENCE of the METHOD
Sodium iodate is separated from other halates and identified by chromatography
the thin layer, and identifies with the oxidation of iodide to form iodine.
2. USED CHEMICALS
All the reagents must be of analytical purity or higher
2.1 standard solutions: aqueous solutions of potassium chlorate, bromate and
potassium iodate 0.2% (m/V) prepared freshly
2.2. mobile phase: 28% of the ammonia solution (m/V) acetone/butanol (60/130/30
V/V/V)
2.3 potassium iodide, aqueous solution of 5% (m/V)
2.4 the starch solution 1 to 5% (m/V)
2.5. Hydrochloric acid, 1 mol/l
3. apparatus and EQUIPMENT
3.1 Ready for cellulose thin-layer chromatography
(thickness of layer 0.25 mm).
3.2 Normal equipment for thin-layer chromatography
4. the procedure
4.1
About 1 g of the sample with water, filter, and dilute to about 10
ml.
4.2.
On the plate (3.1) plated 2 mikrol this solution and 2 mikrol each of
the three standard solutions (2.1).
4.3.
Place the plate in a tank and develop the chromatogram
solvent 2.2 by ascending chromatography about three-quarters of the length of the
the boards.
4.4.
The plate from the tank and allow the solvent to evaporate at ambient
temperature. (Note: it may take up to 2 hours.)
4.5.
The plate with potassium iodide (2.3) and allow about 5
minutes to dry.
4.6.
Spray the plate with starch solution (2.4) and allow to dry for about 5 minutes.
4.7.
Finally spray with hydrochloric acid (2.5).
5. EVALUATION of the
If iodate immediately appears blue spot (the colour may be
be brown or become Brown on standing after a certain time) with an Rf value of 0 to 0.2.
It should be noted that bromates respond immediately with an Rf value
approximately 0.5 to 0.6, chlorates, after about a half hour to respond with a value of
RF 0.7 to 0.8.
(B). THE QUANTITATIVE DETERMINATION OF
1. The DEFINITION of the
The sodium iodate content as determined by this method is expressed in
percentage by mass of sodium iodate.
2. The PRINCIPLE of the
Sodium iodate is dissolved in water and determined by using high-performance
liquid chromatography with two columns in series: with the column with the
reversed phase C18 column and an anion with flux-core wire.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher and, in particular,
must be suitable for the high-performance liquid chromatography (HPLC).
3.1. hydrochloric acid 4 mol/l
3.2. Sodium sulfite in aqueous solution, 5% (m/V)
3.3 the sodium iodate stock solution
Prepare a stock solution by dissolving 50 mg sodium iodate in 100
ml of water.
3.4. Potassium dihydrogen phosphate
3.5 DIPOTASSIUM hydrogen phosphate disodium dihydrate
3.6. Mobile phase for HPLC:
Dissolve 3.88 g potassium dihydrogenorthophosphate (3.4) and 1.19 g
disodium disodného dihydrate (3.5) in 1 litre water.
the pH of this solution is 6.2.
3.7 universal pH indicator paper, pH 1-11
4. apparatus and EQUIPMENT
4.1. Normal laboratory equipment
4.2 the round filter paper, diameter 110 mm, Schleicher and Schuell No.
575, or equivalent
4.3 Device for high performance liquid chromatography equipped with
with a variable wavelength detector
4.4 Columns: length: 120 mm, internal diameter: 4.6 mm, number of: 2 column in the
the series; first column: RP C18 (R) 5 C18 or equivalent; the second
column: Vydac301SB or equivalent
5. the procedure
5.1. Preparation of the sample
5.1.1 the liquid samples (shampoos)
In calibrated 10 ml tubes with ground glass stopper or volumetric
Weigh approximately 1.0 g of the test sample.
Make up the volume with water and mix.
If necessary, filter the solution.
Determine the iodate in the solution by means of HPLC as described in section
5.2.
5.1.2 solid samples (SOAP)
Part of the sample is gently nadrtí and split into the glass cylinder to 100 ml
ground glass stopper, weigh approximately 1.0 g of the test sample. Make up
50 ml with water and shake vigorously 1minutu. Centrifuge and filter
through a filter paper (4.2), or to stand at least overnight.
An undulating mass vigorously and filter through a filter paper
(4.2).
Determine the iodate in the filtrate by means of HPLC as described in section
5.2.
5.2 Chromatography
Flow rate: 1 ml/min.
Detector wavelength (4.3): 210 nm.
Injection volume: 10 mikrol.
Measured variable: peak area.
5.3 Calibration
In a 50-ml volumetric flasks transfer by pipette 1.0, 2.0, 5.0, 10.0 and 20.0 ml of the
sodium iodate stock solution (3.3). The volumes make up to the mark and
mix.
Solutions contain 0.01, 0.02, 0.05, 0.1, and 0.20 mg iodate
sodium in 1 ml.
Inject 10 to column mikrol of each standard and recorded
the chromatogram. Determine the peak area for iodate and plot a calibration
the curve of the peak area to the sodium iodate concentration.
6. The CALCULATION of the
The sodium iodate content expressed as a percentage by mass (m/m)
calculated from the formula:
VC
% (m/m) of sodium iodate =-----
10 m
where
m = mass of the test sample (5.1) in grams,
V = the total volume of the sample solution obtained in 5.1, in millilitres,
c = concentration of sodium iodate in mg per millilitre, obtained from the calibration
curve (5.3).
7. REPEATABILITY
For products containing about 0.1% (m/m) of sodium iodate variation
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.002%.
8. CONFIRMATION of the
8.1 the principle
In an acidified solution of a cosmetic product, iodate (IO3-)
reduced to iodide (I-) by sulphite and the resulting solution is analysed
high-performance liquid chromatography. If a peak having a retention
the time corresponding to the retention time of iodate reaction, sulphite
disappear, it was most likely the iodate.
8.2 Procedure
Erlenmeyer flask transfer by pipette 5 ml of the sample prepared
According to section 5.1.
the pH of the solution is adjusted with hydrochloric acid (3.1) to a value of 3 or
lower; universal indicator paper (3.7).
Add three drops of sodium sulphite solution (3.2) and
mix.
10 mikrol the solution is injected into the column liquid chromatography
(4.3).
The resulting chromatogram is compared with chromatogramem recorded for the
the same sample referred to in section 5.
30. The qualitative and quantitative DETERMINATION of SILVER NITRATE in
COSMETIC PRODUCTS
And qualitative determination
1. scope and field of application
This method describes the qualitative determination of silver nitrate
expressed as silver in cosmetic products based on water.
2. The principle of the
Qualitative determination of silver is carried out through the
the characteristic white precipitate with chloridovými ions.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. hydrochloric acid solution, 2 mol/l
3.2 ammonium hydroxide aqueous solution:
concentrated ammonium hydroxide solution (d204 = 0.88 g/ml) diluted
the same quantity of water and mix thoroughly.
3.3. nitric acid solution, 2 mol/l
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2 the Centrifuge
5. the procedure
5.1
To approximately 1 g of sample in a centrifuge tube, add Dropwise 2 m
hydrochloric acid (3.1), until the total precipitation;
mix and centrifuge.
5.2.
The supernatant liquid and wash the precipitate five drops
the cold water. Washings.
5.3.
In the centrifuge tube to the precipitate add the same volume of water. For
stirring is heated to a boil.
5.4.
Centrifuge hot; the supernatant liquid.
5.5
To the precipitate add a few drops of ammonia solution (3.2); mix
and centrifuge.
5.6 to one drop of the liquid above the precipitate on a glass slide Add
a few drops of 2 mol/l nitric acid (3.3).
3.5 a white precipitate indicates the presence of silver.
(B). The quantitative determination of
1. scope and field of application
This method is suitable for the determination of silver nitrate,
expressed as silver in cosmetic products intended to dye
eyelashes and eyebrows.
2. The principle of the
Silver is in the product by atomic absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Nitric acid solution, 0.02 mol/l solution
3.2 standard solutions of silver
3.2.1
Stock silver standard solution, 1 000 mikrog/ml in 0.5 mol/l
nitric acid solution (' SpectrosoL ' or equivalent)
3.2.2.
Silver standard solution, 100 mikrog/ml 10 ml of the stock standard
Silver solution (3.2.1) into a volumetric flask transfer by pipette on the 100 ml.
Make up to volume with 0.02 mol/l solution of nitric acid and mix.
This standard solution is prepared fresh and stored in the
glass bottle of dark glass.
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. Atomic Absorption Spectrometer fitted with a silver hollow
cathode.
5. the procedure
5.1. Preparation of the sample
Weigh approximately 0.1 g (m gram) of an homogenous sample. This quantity
volumetric flask, make up to one litre of volume with 0.02
mol/l nitric acid solution (3.1) and mix.
5.2 Conditions for atomic absorption spectrometry
Flame: vzduchacetylen
Wavelength: 338.3 nm
Background correction: Yes
Fuel requirements: poor; for maximum absorbance is needed
optimization of burner height and fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 100 ml volumetric flasks transfer by pipette 1.0; 2.0; 3.0; 4.0 and
5.0 ml of the silver standard solution (3.2.2). Make up each flask
volume with 0.02 mol/l nitric acid solution (3.1) and
mix. These solutions contain 1.0; 2.0; 3.0; 4.0 and 5.0 mikrog silver
per millilitre.
5.3.2.
Measure the absorbance of a 0.02 mol/l nitric acid solution (3.1) and
the value obtained as the zero silver concentration for the
the calibration curve. Measure the absorbance of each standard solution
Silver (5.3.1). Plot a calibration curve relating depending
the absorbance values for the concentration of silver.
5.4. The quantitative determination of
Measure the absorbance of the sample solution (5.1). From the calibration curve read off the
the concentration of silver corresponding to the value of the absorbence measured for the solution
sample.
6. The calculation of the
The silver nitrate content of the sample expressed in weight
percentages (% m/m) is calculated by the formula
1.5748 x (c)
% (m/m) of silver nitrate =---------------------
10 x m
where
m = mass of the test sample (5.1) in grams,
c = concentration of silver in the sample solution (5.1) and decreases the
from the calibration curve in mikrog/ml.
7. Repeatability
For products containing about 4% (m/m) of silver nitrate, the difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.05%.
31. The qualitative and quantitative DETERMINATION of the CONTENT of SELENIUM DISULPHIDE
IN ANTI-DANDRUFF SHAMPOOS
And qualitative determination
1. scope and field of application
This method describes the qualitative determination of selenium disulphide
as selenium in anti-dandruff shampoos.
2. The principle of the
Qualitative determination of Selenium is identified by the
the characteristic yellow to Orange colouration in the response with the
urea and potassium iodide.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. nitric acid, concentrated (d204 = 1.42 g/ml)
3.2 Urea
3.3. potassium iodide, 10% (m/V) solution: 10 g of potassium iodide,
dissolved in 100 ml of water.
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. digestion tube, 100 ml flask
4.3 Digestion device with vyhřívacím block
4.4. Filter paper (Whatman No 42 or equivalent) or a membrane
filter (pore size 0.45 mikrom)
5. the procedure
5.1
To approximately 1 g of shampoo in a digestion tube (4.2) add 2.5 ml
concentrated nitric acid (3.1) and in the digesčním device with
vyhřívacím block (4.3) at 150 ° C for 30 minutes.
5.2.
Dilute the digested sample to 25 ml water, filter through
filter paper or through a membrane filter (0.45 mikrom) (4.4).
5.3.
To 2.5 ml of the filtrate add 5 ml water, 2.5 g urea (3.2) and boil.
Cool and add 1 ml of potassium iodide solution (3.3).
5.4.
Yellow to orange colour which darkens rapidly on standing indicates the
the presence of selenium.
(B). The quantitative determination of
1. scope and field of application
This method is suitable for the quantitative determination of selenium disulphide
as selenium in anti-dandruff shampoos containing not more than 4.5% (m/m)
Selenium sulfide.
2. The principle of the
The sample is digested with nitric acid and selenium in the resulting extract
by atomic absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. nitric acid, concentrated (d204 = 1.42 g/ml)
3.2. Nitric acid solution, 5% (V/V) solution:
in 500 ml water in a beaker, stirring continuously, add 50 ml
concentrated nitric acid (3.1). Transfer this solution to a
a 1 litre volumetric flask and make up to the mark with water.
3.3. stock selenium standard solution, 1 000 mikrog/ml in 0.5 mol/l
nitric acid solution (' SpectrosoL ' or equivalent)
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. digestion tube, 100 ml flask
4.3 Digestion device with vyhřívacím block
4.4. Filter paper (Whatman No 42 or equivalent) or a membrane
filter, 0.45 mikrom
4.5. atomic absorption spectrophotometer equipped with a lamp with selenovou
hollow-cathode lamp
5. the procedure
5.1. Preparation of the sample
5.1.1.
Into a digestion tube (4.2) weigh about 0.2 g (m gram) of an homogenous
sample of shampoo exactly.
5.1.2.
Add 5 ml of concentrated nitric acid (3.1) and one
hour in the digesčním device with vyhřívacím block (4.3) at 150
St. (C).
5.1.3.
Allow to cool and dilute with water to 100 ml. Filter
through a filter paper or a membrane filter (0.45 mikrom) (4.4) and
retain the filtered solution for quantitative determination.
5.2 Conditions for atomic absorption spectrometry
Flame: vzduchacetylen
Wavelength: 196.0 nm
Background correction: Yes
Fuel condition:
the poor; for maximum absorbance is necessary optimization of burner height and
fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 100 ml volumetric flasks transfer by pipette 1.0; 2.0; 3.0; 4.0 and
5.0 ml of the stock selenium standard solution (3.3). Each flask
make up to volume with 5% (m/m) of the nitric acid solution (3.1) and
mix. These solutions contain 10, 20, 30, 40 and 50 mikrog selenium on
millilitre.
5.3.2.
Measure the absorbance of a 5% solution of nitric acid (3.1) and thus obtained
value is used as the zero selenium concentration for the calibration
the curve. Measure the absorbance of each selenium standard solution
(5.3.1) plot a calibration curve relating dependent values
absorbance concentration of selenium.
5.4. The quantitative determination of
Measure the absorbance of the sample solution (5.1.3). From the calibration curve
subtracts the Selenium concentration corresponding to the value of the absorbence measured for
the sample solution.
6. The calculation of the
The contents of selenium disulphide in the sample, in percentage by mass (% m/m)
shall be calculated according to the formula:
1.812 × c
% (m/m) of selenium disulphide =----------
100 x m
where
m = mass of the test sample (5.1) in grams,
c = concentration of selenium in the sample solution (5.1.3) deducted
from the calibration curve in mikrog/ml.
7. Repeatability
For products that contain about 1% (m/m) of selenium disulphide difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.05%.
32. The QUANTITATIVE DETERMINATION of SOLUBLE BARIUM and strontium in pigments
IN THE FORM OF SALTS OR COMPLEXES
And Quantitative determination of soluble barium
1. scope and field of application
This method describes the procedure for the extraction and quantitative determination of
soluble barium from pigments in the form of salts or complexes.
2. The principle of the
The pigment is extracted with 0, 07 m hydrochloric acid solution under
defined conditions and the amount of barium in the extractant determined by Atomic
absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Ethanol, absolute
3.2. Hydrochloric acid solution, 0.07 mol/l solution
3.3. hydrochloric acid, 0.5 mol/l solution
3.4. potassium chloride, 8% (m/V) solution:
16 g of potassium chloride in 200 ml, dissolve 0, 07 m acid solution
hydrochloric acid (3.2).
3.5. barium standard solutions
3.5.1. stock barium standard solution, 1 000 mikrog/ml in 0.5 mol/l
nitric acid solution (' SpectrosoL ' or equivalent)
3.5.2. Barium standard solution, 200 mikrog/ml:
20.0 ml of the stock barium standard solution (3.5.1) into the pipette
volumetric flasks, 100 ml. Make up to volume with 0.07 mol/l solution
hydrochloric acid (3.2) and mix.
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2 pH meter with an accuracy of +/-0,02
4.3 the Shaker with swirl
4.4. membrane filter with a pore size of 0.45 mikrom
4.5. atomic absorption spectrophotometer equipped with a lamp with a barium meal
hollow-cathode lamp
5. the procedure
5.1. Preparation of the sample
5.1.1.
Erlenmeyer flask, weigh about 0.5 g of pigment exactly (m grams).
For the effective mixing is appropriate to use the flask on a minimum volume of 150
ml.
5.1.2.
Add by pipette 1.0 ml of ethanol (3.1) and rotate the circle so that the
ensure thorough wetting of the pigment. From a burette, add exactly the quantity of 0.07
mol/l hydrochloric acid solution (3.2), which is necessary to
that was achieved by the ratio of the volume of acid to the weight of pigment exactly 50
millilitres per gram. The total volume of extractant including the ethanol be marked "in
ML ". In order to achieve a thorough mixing of the contents of the flask for 5 seconds
circling.
5.1.3.
pH meter (4.2) measure the pH of the resultant suspension and, if over a value of 1.5,
Add Dropwise 0.5 mol/l hydrochloric acid solution (3.3),
until it reaches the value of 1.4 to 1.5.
5.1.4
Stopper the flask and shake in a Shaker for 60 minutes with a circular
movement (4.3). Shaker must have sufficient speed to
foam. Filter through a membrane filter (4.4) 0.45 mikrom and filtrate
accumulated. The extract is not centrifuge before zfiltrováním. 5.0 ml of the filtrate
transfer to a volumetric flask of 50 ml; make up to volume with 0.07 m/l
hydrochloric acid (3.2) and mix. This solution is also
used to determine the content of strontium (part B).
5.1.5.
In a 100 ml volumetric flask transfer by pipette 5.0 ml of the solution of chloride
(3.4) and an aliquot (WBa ml) of the diluted filtrate
(5.1.4) to give an expected concentration of between 3 to 10 mikrog barium on
millilitre. (An aliquot of 10 ml should be a default
quantity.) Make up to volume with 0.07 mol/l acid solution
hydrochloric acid (3.2) and mix.
5.1.6.
Barium concentration of the solution (5.1.5) by atomic absorption
spectrometry on the same day.
5.2 Conditions for atomic absorption spectrometry
Flame: dusnýacetylen
Wavelength: 553.5 nm
Background correction: no
Fuel condition:
the poor; for maximum absorbance is necessary optimization of burner height and
fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 100 ml volumetric flasks transfer by pipette 1.0; 2.0; 3.0; 4.0 and
5.0 ml of the barium standard solution (3.5.2). To each flask transfer by pipette
5.0 ml potassium chloride solution (3.4). Make up to volume with 0.07 m/l
hydrochloric acid solution (3.2) and mix. These
solutions contain 2.0; 4.0; 6.0; 8.0 and 10.0 mikrog barium per millilitre.
Similarly, prepare a blank test with the standard solution
barium.
5.3.2.
Measure the absorbance of the solution (5.3.1) and use the value obtained with the
used as the zero barium concentration for the calibration curve. Measure the
absorbance of each barium standard solution (5.3.1). Plot the
plot a calibration curve relating absorbance values to the concentration
barium.
5.4 determination of the
Measure the absorbance of the sample solution (5.1.5). From the calibration curve
subtracts the barium concentration corresponding to the value of the absorbence measured for
the sample solution.
6. The calculation of the
Soluble barium content (% m/m) of the pigment is given by the formula
(C) the x in
% (m/m) of soluble barium =--------------
10 WBa x m
where
m = mass of the sample taken for analysis (5.1.1), in micrograms,
(c) = barium concentration of the sample solution (5.1.5) deducted
from the calibration curve in mikrog/ml,
V = total volume of extractant in millilitres (5.1.2),
WBA = volume of extract collected in accordance with section 5.1.5 in ml.
7. Repeatability
For products containing about 2% (m/m) of soluble barium is for this
the best estimate of the repeatability method 0.3%.
8. Notes
8.1.
Under certain conditions the barium absorbance can be enhanced by the presence of
calcium. This can be prevented by the addition of magnesium ion at a concentration of 5 g/l
(see ' Magnesium as modifier for the determination of barium by flame
Atomic emission spectrometry ". Jerrow, m. et al., Analytical
Proceedings, 1991, 28, 40.).
8.2.
The use of inductively-coupled plasma-optical
emission spectrometry is permitted as an alternative to flame Atomic
absorption spectrometry.
(B). The quantitative determination of soluble strontium
1. scope and field of application
This method describes the procedure for the extraction and quantitative determination of
soluble strontium from pigments in the form of salts or complexes.
2. The principle of the
The pigment is extracted with 0, 07 m hydrochloric acid solution under
defined conditions and the amount of strontium in the extractant determined by
atomic absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Ethanol, absolute
3.2. Hydrochloric acid solution, 0.07 mol/l solution
3.3. potassium chloride, 8% (m/V) solution:
16 g of potassium chloride in 200 ml, dissolve 0.07 mol/l solution
hydrochloric acid (3.2).
3.4. strontium standard solutions
3.4.1.
Stock strontium standard solution, 1 000 mikrog/ml in 0.5 mol/l solution
nitric acid solution (' SpectrosoL ' or equivalent)
3.4.2
Strontium standard solution, 100 mikrog/ml: 10.0 ml of the stock
barium standard solution (3.4.1) into a volumetric flask transfer by pipette to
100 ml. Make up to volume with 0.07 mol/l hydrochloric acid solution
(3.2) and mix.
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. membrane filter with a pore size of 0.45 mikrom
4.3. atomic absorption spectrophotometer equipped with a lamp with stronciovou
hollow-cathode lamp
5. the procedure
5.1. Preparation of the sample
For the quantitative determination of soluble strontium solution
prepared in section and 5.1.4.
5.1.1.
In a 100 ml volumetric flask transfer by pipette 5.0 ml of the solution of chloride
(3.3) and an aliquot (WSr ml) of the diluted filtrate (and
5.1.4), estimated to be 2 to 5 of the strontium concentration mikrog/ml.
(An aliquot of 10 ml should be a default quantity.) Make up
to volume with 0.07 mol/l hydrochloric acid solution (3.2) and
mix.
5.1.2.
Strontium concentration in the solution (5.1.1) by atomic absorption
spectrometry on the same day.
5.2 Conditions for atomic absorption spectrometry
Flame: dusnýacetylen
Wavelength: 460.7 nm
Background correction: no
Fuel condition:
the poor; for maximum absorbance is necessary optimization of burner height and
fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 100 ml volumetric flasks transfer by pipette 1.0; 2.0; 3.0; 4.0 and
5.0 ml of the strontium standard solution (3.4.2). To each flask,
transfer by pipette 5.0 ml potassium chloride solution (3.3). Make up to the mark with
0.07 mol/l hydrochloric acid solution (3.2) and mix.
These solutions contain 1.0; 2.0; 4.0 and 5.0 mikrog strontium per millilitre.
Similarly, prepare a blank test with the standard solution
strontium.
5.3.2.
Measure the absorbance of the solution (5.3.1) and use the value obtained with the
used as the zero strontium concentration for the calibration curve. Measure the
the absorbance of each strontium calibration standard (5.3.1). Plot
a calibration curve relating absorbance values to
the concentration of strontium.
5.4 determination of the
Measure the absorbance of the sample solution (5.1.1). From the calibration curve
subtracts the strontium concentration corresponding to the value of the absorbence measured
for the sample solution.
6. The calculation of the
Soluble strontium content (% m/m) of the pigment is given by the formula
(C) the x in
% (m/m) of soluble strontium =--------------
10 WSr x m
where
m = mass of the test sample (and 5.1.1) in (g),
c = strontium concentration in the sample solution (5.1.1) deducted
from the calibration curve in mikrog/ml,
V = the total volume of the extract (and 5.1.2) in ml
WSR = volume of extract collected in accordance with section 5.1.1 in ml.
7. Repeatability
For products containing about 0.6% (m/m) of soluble barium is for this
the best estimate of the repeatability method 0.09%.
8. the Note
The use of inductively-coupled plasma-optical
emission spectrometry is permitted as an alternative to flame Atomic
absorption spectrometry.
33. The qualitative and quantitative DETERMINATION of BENZYL ALCOHOL
And qualitative determination
1. scope and field of application
This method describes the qualitative determination of benzyl alcohol in the
cosmetic products.
2. The principle of the
Qualitative determination of benzyl alcohol, chromatography
a thin layer of silica gel.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Benzyl alcohol
3.2. Chloroform
3.3. Ethanol, absolute
3.4 nPentan
3.5 mobile phase: ethyl ether
3.6. standard solution of benzyl alcohol:
In a 100 ml volumetric flask, weigh 0.1 g of benzyl alcohol (3.1), make up to
the mark with ethanol (3.3) and mix.
2.3 plates for thin-layer chromatography, glass, 100 x 200 mm
or 200 x 200 mm, coated with a layer of silica gel 60 F254
0.25 mm.
3.8. visualizing agent:
12molybdofosforečná acid, 10% (m/V) solution in ethanol (3.3).
4. apparatus and equipment
4.1 Normal equipment for thin-layer chromatography
4.2 saturate with two grooves, the outer dimensions of approximately
80 mm x 230 mm x 240 mm
4.3. Chromatography paper: Whatman, or equivalent
4.4. Ultra-violet lamp, wavelength 254 nm.
5. the procedure
5.1. Preparation of the sample
Into volumetric flasks 10 ml, weigh 1.0 g of the product to be
parsed. Add 3 ml of chloroform (3.2) and shake it,
until the resource has dispersed. Make up to volume with ethanol (3.3) and
shake thoroughly until the emergence of a clear or almost clear solution.
5.2. Thin-layer chromatography
5.2.1.
Saturate with (4.2) npentanem as follows: party Chambers
adjacent to the back through with chromatography paper (4.3)
so that the bottom edge of the paper is in the trough. The rear Groove is filled with 25 ml
npentanu (3.4) the spill-over of this solution over the surface of the chromatographic
the paper. Immediately fit the lid and leave to stand for 15 minutes.
5.2.2.
At suitable points on the start line of the chromatographic plates
thin-layer chromatography is spotted 10 mikrol of the sample solution (5.1)
and 10 mikrol benzyl alcohol standard solution (3.6). Allow to
dry.
5.2.3.
To the front of the chute pipette 10 ml of diethyl ether (3.5) and
into the same Groove immediately place the plate (5.2.2). Once again, quickly
deploys the lid of the tank and the Board develops to a height of 150 mm. Plate from the
chromatography tank and allow to dry at room temperature.
5.2.4
The plate (5.2.3) under ultra-violet light and mark the position of the
the violet spots. The plate is the visualizing agent (3.8) and then
heated for 15 minutes at 120 ° C. Benzyl alcohol appears as dark blue
the stain.
5.2.5
Calculate the Rf value obtained using the standard solution
benzyl alcohol. A dark blue spot with the same Rf value obtained from the
the sample solution indicates the presence of benzyl alcohol.
Detection limit: 0.1 mikrog benzyl alcohol.
(B). The quantitative determination of
1. scope and field of application
This method is described in the quantitative determination of the content of benzyl alcohol
in cosmetic products.
2. The definition of the
The amount of benzyl alcohol determined by this method is expressed in
percentage by mass (% m/m).
3. The principle of the
The sample is extracted with methanol and the amount of benzyl alcohol in the extract
be determined by high-performance liquid chromatography (HPLC).
4. CHEMICALS USED
All the reagents must be of analytical purity or higher and according to
needs must be suitable for HPLC.
4.1 Methanol
4.2 4Ethoxyfenol
4.3. Benzyl alcohol
4.4. mobile phase: methanol (4.1)/water (45:55; IN/IN)
4.5. benzyl alcohol stock solution:
in a 100 ml volumetric flask, weigh accurately approximately 0.1 g of benzyl alcohol
(4.3) make up to the mark with methanol (4.1) and mix thoroughly.
4.6. Internal standard stock solution:
in a 100 ml volumetric flask, weigh about 0.1 g of 4-ethoxyphenol (4.2)
exactly. Make up to the mark with methanol (4.1) and mix thoroughly.
4.7 standard solutions:
In a series of 25-ml volumetric flasks transfer by pipette stock solution
benzyl alcohol (4.5) and internal standard stock solution (4.6) according to the
in the table below. Make up to the mark with methanol (4.1) and mix thoroughly.
-----------------------------------------------------------------
The standard concentration of benzyl alcohol Concentration
solution of 4-ethoxyphenol
the number of additions mikrog/ml number of mikrog/ml
ml (4.5) (*) (*) added
ml (4.6)
-----------------------------------------------------------------
And 0.5 2.0 80 20
(II) 1.0 2.0 40 80
(III) 2.0 2.0 80 80
IV 2.0 3.0 120 80
In 200 4.0 2.0 80
-----------------------------------------------------------------
(*) These values are given as an indication and correspond to the
concentrations of the standard solutions prepared from solutions
benzyl alcohol (4.5) and the 4-ethoxyphenol (4.6), which contain exactly
0.1% (m/V) benzyl alcohol and exactly 0.1% (m/V) 4-ethoxyphenol.
5. apparatus and equipment
5.1. Normal laboratory equipment
5.2 equipment for high-performance liquid chromatography with UV
with a variable wavelength detector and with injection loop
Volume 10 mikrol
5.3. analytical column: stainless steel: 250 mm x 4.6 mm
Spherisorb ODS particle size 5 mikrom or equivalent
filling.
5.4. water bath
5.5. ultrasonic bath
5.6. centrifuge
5.7. Centrifuge tube 15 ml
6. the procedure
6.1. Preparation of the sample
6.1.1.
In the centrifuge tube (5.7) weigh about 0.1 g (m gram) of sample
exactly and add 5 ml of methanol (4.1).
6.1.2.
Heat for 10 minutes in a water bath (5.4) maintained at a temperature
50 ° C, and then is placed in an ultrasonic bath (5.5), where the leaves to
sample is thoroughly dispersed.
6.1.3
Cool and then centrifuged for 5 minutes at 3 500 rpm.
6.1.4.
The supernatant is transferred to a volumetric flask, 25 ml.
6.1.5
The rest of the sample with a further 5 ml methanol (4.1). Extracts
merges in 25 ml volumetric flask.
6.1.6
Volumetric flask, 25 ml odpipetují 2.0 ml of the stock solution
the internal standard (4.6). Make up to the mark with methanol (4.1) and
mix. This solution is used for chromatography described in point
6.4.
6.2 the Chromatography
6.2.1.
The usual way is set for high-performance liquid
chromatography (3.2). The flow rate of the mobile phase (4.4)
to 2.0 ml/min.
6.2.2.
Wavelength UV detector (5.2) to 210 nm.
6.3 Calibration
6.3.1.
Inject 10 mikrol each of the standard solutions
benzyl alcohol (4.7) and measure the areas of the benzyl alcohol and the 4-
ethoxyphenol peaks.
6.3.2.
For each benzyl alcohol standard solution (4.7) calculate the ratio
of benzyl alcohol to 4-ethoxyphenol desktop. The calibration curve
by plotting these ratios on the y axis and the corresponding concentrations of
benzyl alcohol in mikrog/ml on the horizontal axis.
6.4 quantitative determination
6.4.1
On the column inject 10 mikrol of the sample solution (6.1.6) and measure the
the areas of the benzyl alcohol 4-ethoxyphenol. Calculate the peak area ratio
benzyl alcohol and 4-ethoxyphenol. Repeat this procedure with the other with
After 10 mikrol aliquots of the sample solution, as long as they are given
identical results.
6.4.2
From the calibration curve (6.3.2) read off the concentration of benzyl alcohol
corresponding to the peak area ratio of benzyl alcohol to 4-ethoxyphenol and.
7. The calculation of the
Benzyl alcohol content of the sample, in percentage by mass, is calculated
According to the formula:
(c)
% (m/m) benzylakoholu =---------
400 x m
where
m = mass of test sample in g (6.1.1),
c = concentration of benzyl alcohol in the sample solution (6.1.1)
in mikrog/ml, obtained from the calibration curve.
8. Repeatability
For products that contain about 1% (m/m) of benzyl alcohol, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.10%.
34. qualitative DETERMINATION of ZIRCONIUM and the QUANTITATIVE DETERMINATION of zirconium
ALUMINIUM AND CHLORINE IN NON-AEROSOL ANTIPERSPIRANTS
This method has five stages:
And qualitative determination of zirconium
(B). The quantitative determination of zirconium
C. Quantitative determination of aluminium
(D). Quantitative determination of chlorine
(E) calculation of the proportion of content. aluminium atoms to zirconium atoms, and content
calculation of the relative content of aluminium plus zirconium atoms to chlorine atoms content.
And qualitative determination of zirconium
1. scope and field of application
This method describes the qualitative determination of zirconium in
non-aerosol antiperspirants. There are described methods appropriate for
qualitative determination of aluminium zirconium chloride hydroxide complexes of aluminium and zirconium
Alxzr (OH) yClZ.
2. The principle of the
Qualitative determination of zirconium is carried out through the creation of
the distinctive red-violet precipitate reaction with alizarinovou
Red with in strongly acidic media.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. hydrochloric acid, concentrated (d20 = 1, 18 g/ml)
3.2 the solution with alizarinu (CI 58005:2% (m/V) sodium alizarinsulfonátu
in the water)
4. apparatus and equipment
4.1. Normal laboratory equipment
5. the procedure
5.1
To approximately 1 g of sample in a test tube add 2 ml of water. Stopper and
shake vigorously.
5.2.
Add three drops of Alizarin red S solution (3.2) and 2 ml
concentrated hydrochloric acid (3.1). Stopper and shake vigorously.
5.3.
Leave for approximately 2 minutes.
5.4.
The reddish colour of the supernatant and precipitate indicates the presence of
zirconium.
(B). The quantitative determination of zirconium
1. scope and field of application
This method is suitable for the quantitative determination of zirconium in
aluminium zirconium chloride hydroxide complexes of aluminium and zirconium, up to the maximum concentration of
zirconium 5.4% (m/m) in non-aerosol antiperspirants.
2. The principle of the
Zirconium is extracted from the resource in an acidic environment and quantitative
the determination shall be made by flame atomic absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. hydrochloric acid, concentrated (d20 = 1.18 g/ml)
3.2. hydrochloric acid solution, 10% (V/V):
to 500 ml of water in a beaker, stirring continuously, add 100 ml
concentrated hydrochloric acid (3.1). Transfer this solution to a
1 litre volumetric flask and make up to the mark with water.
3.3. stock zirconium standard solution, 1000 mikrog/ml in 0.5 mol/l
hydrochloric acid solution (' SpectrosoL ' or equivalent)
3.4. Aluminium chloride (hydrated) [Alcl3.6h2o]:
Agent: hexahydrátu 22.6 g of aluminium chloride is dissolved in 250 ml
10% (V/V) hydrochloric acid solution (3.2).
3.5. Ammonium chloride:
Agent: 5.0 g of ammonium chloride in 250 ml dissolve the 10% (V/V)
hydrochloric acid solution (3.2).
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. Heater with magnetic stirrer
4.3. Filter paper (Whatman No 41 or equivalent)
4.4. atomic absorption spectrophotometer equipped with a lamp with zirkoniovou
hollow-cathode lamp
5. the procedure
5.1. Preparation of the sample
5.1.1.
150 ml beaker weigh approximately 1.0 g (m gram) of an homogenous sample
resource exactly. Add 40 ml of water and 10 ml of the concentrated acid
hydrochloric (3.1).
5.1.2.
Place the beaker on a hot plate with a magnetic stirrer (4.2). Turns on
stirring and heat to boiling. To prevent rapid drying
the beaker is placed on the watch-glass. Boil for 5 minutes, remove the beaker from the
heating plate and cool to room temperature.
5.1.3.
Using the filter paper (4.3) to the contents of the beaker into
volumetric flasks, 100 ml. Beaker twice with 10 ml of water and
rinsing water after filtration added to the flask. Make up to the mark with
water and mix. This solution is also used for quantitative
determination of aluminium (part C).
5.1.4
Into a 50 ml volumetric flask transfer by pipette 20.00 ml of the sample solution
(5.1.1), 5.00 ml of the aluminium chloride reagent (3.4) and 5.00 ml reagent
ammonium chloride (3.5). Make up to the mark with 10% (V/V) acid solution
hydrochloric acid and mix.
5.2 conditions for atomic absorption spectrometry
Flame: nitrous oxide/acetylene
Wavelength: 360.1 nm
Background correction: no
Fuel condition:
rich; for maximum absorbance is necessary optimization of burner height and
fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 50 ml volumetric flasks, transfer by pipette 5.00; 10.00; 15.00;
20.0 and 25.00 ml of the stock zirconium standard solution (3.3). To each
volumetric flask transfer by pipette 5.00 ml of the aluminium chloride reagent (3.4) and
5.00 ml of the ammonium chloride reagent (3.5). Make up to the mark with 10% (V/V)
hydrochloric acid (3.2) and mix. These solutions contain
100, 200, 300, 400 and 500 mikrog zirconium per millilitre.
Similarly, prepare a blank test omitting the zirconium standard solution.
5.3.2.
Measure the absorbance of the solution (5.3.1) and use the value obtained
as the zero zirconium concentration for the calibration curve. Measure the
absorbance of each zirconium calibration standard (5.3.1). Plot the
plot a calibration curve relating absorbance values to the concentration
zirconium.
5.4. The quantitative determination of
Measure the absorbance of the sample solution (5.1.4). From the calibration curve
read off the concentration of zirconium corresponding to the value of the absorbance measured
for the sample solution.
6. The calculation of the
Zirconium content of the sample, in percentage by mass, is calculated
According to the formula:
(c)
% (m/m) of zirconium =--------
40 x m
where
m = mass of the sample taken for analysis (5.1.1), in micrograms,
c = concentration of zirconium in the sample solution (5.1.4) mikrog/ml,
obtained from the calibration curve.
7. Repeatability
For products containing about 3% (m/m) of zirconium, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.10%.
8. the Note
The use of inductively-coupled plasma-optical
emission spectrometry is permitted as an alternative to flame Atomic
absorption spectrometry.
C. Quantitative determination of aluminium
1. scope and field of application
This method is suitable for the quantitative determination of aluminium in
aluminium zirconium chloride hydroxide complexes of aluminium and zirconium to the concentration of aluminium
12% (m/m) in non-aerosol antiperspirants.
2. The principle of the
Aluminium is extracted from the resource in an acidic environment and quantitative
the determination shall be made by flame atomic absorption spectrometry.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. hydrochloric acid, concentrated (d20 = 1.18 g/ml)
3.2. Hydrochloric acid solution, 1% (V/V) solution:
to 500 ml of water in a beaker, stirring continuously, add 10 ml concentrated
hydrochloric acid (3.1). Transfer this solution into the volumetric flask
1 litre, and make up to the mark with water.
3.3. stock aluminium standard solution:
1 000 mikrog/ml in 0,5 m nitric acid solution (' SpectrosoL ' or
equivalent)
3.4 Reagent potassium chloride reagent:
10.0 g of potassium chloride in 250 ml of 1% acid
hydrochloric acid (3.2).
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. atomic absorption spectrophotometer equipped with an aluminum lamp with
hollow-cathode lamp
5. the procedure
5.1. Preparation of the sample
For the quantitative determination of aluminium prepared in solution
part (B) 5.1.3.
5.1.1.
In a 100 ml volumetric flask transfer by pipette 5.00 ml of the sample solution (B
5.1.3) and 10.00 ml of the potassium chloride reagent (3.4). Make up to
the mark with 1% solution of hydrochloric acid (3.2) and mix.
5.2 conditions for atomic absorption spectrometry
Flame: dusnýacetylen
Wavelength: 309.3 nm
Background correction: no
Fuel condition:
rich; for maximum absorbance is necessary optimization of burner height and
fuel conditions.
5.3 Calibration
5.3.1.
Into a series of 100 ml volumetric flasks, transfer by pipette 1.00; 2.00; 3.00; 4.00
and 5.00 ml stock aluminium standard solution (3.3). To each
volumetric flask transfer by pipette 10.00 ml of the potassium chloride
(3.4), make up to the mark with 1% solution of hydrochloric acid (3.2) and
mix. These solutions contain 10, 20, 30, 40 and 50 mikrog aluminum
per millilitre.
Similarly, prepare a blank test omitting the aluminium standard solution.
5.3.2.
Measure the absorbance of the solution (5.3.1) and use the value obtained with the
used as the zero aluminium concentration for the calibration curve. Measure the
absorbance of each aluminium standard solution. The calibration
curve relating absorbance values to the concentration of aluminium.
5.4. The quantitative determination of
Measure the absorbance of the sample solution (5.1.1). From the calibration curve
read off the concentration corresponding to the value of the absorbence measured aluminum for
the sample solution.
6. The calculation of the
Aluminium content of the sample, expressed as percentage by mass is calculated
According to the formula:
(c)
% (m/m) of aluminium =----
5 x m
where
m = mass of the test sample (5.1.1), in micrograms,
c = concentration of aluminium in the sample solution (5.1.1) in mikrog/ml,
obtained from the calibration curve.
7. Repeatability
For products containing about 3.5% (m/m) of aluminium, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.10%.
8. the Note
The use of inductively-coupled plasma-optical
emission spectrometry is permitted as an alternative to flame Atomic
absorption spectrometry.
(D). Quantitative determination of chlorine
1. scope and field of application
This method is suitable for the quantitative determination of chlorine present in the
the form of chloride ion in aluminium zirconium chloride hydroxide complexes of aluminium and zirconium
in non-aerosol antiperspirants.
2. The principle of the
The content of chloride ion in the resource is determined by potentiometric
by titration with a standard solution of silver nitrate.
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. nitric acid, concentrated (d20 = 1.42 g/ml)
3.2. Nitric acid solution, 5% (V/V) solution:
to 250 ml water in a beaker, stirring continuously, add 25 ml
concentrated nitric acid (3.1). This solution is transferred to the
500 ml volumetric flask and make up to the mark with water.
3.3 Acetone
3.4. silver nitrate, 0.1 mol/l standard solution (' AnalaR ' or
equivalent)
4. apparatus and equipment
4.1. Normal laboratory equipment
4.2. Heater with magnetic stirrer
4.3. Silver electrode
4.4. calomel electrode referentní
4.5 pH/Millivolt meter suitable for potentiometric titration
5. the procedure
5.1. Preparation of the sample
5.1.1.
250 ml beaker weigh approximately 1.0 g (m gram) of an homogenous
sample resource exactly. Add 80 ml of water and 20 ml of solution 5%
nitric acid (3.2).
5.1.2.
Place the beaker on a hot plate with a magnetic stirrer (4.2). Turns on
stirring and heat to boiling. To prevent rapid drying
the beaker is placed on the watch-glass. Boil for 5 minutes, remove the beaker from the
heating plate and cool to room temperature.
5.1.3.
Add 10 ml of acetone (3.3), dip electrodes (4.3 and 4.4) below
surface of solution and commence stirring. Potentiometric titration is performed
0.1 mol/l silver nitrate solution (3.4) and construct a
differential curve to determine the equivalence point (in ml).
6. The calculation of the
The content of chlorine in the sample, expressed as a percentage by mass,
calculated according to the formula:
0.3545 x in
% (m/m) of chlorine =-----------
m
where
m = mass of the sample anylyzovaného (5.1.1) in (g),
V = volume of 0.1 mol/l solution of silver nitrate, in millilitres,
used for the titration in the equivalence point.
7. Repeatability
For products containing about 4% (m/m) of chlorine, the difference between the results of the
two parallel on the same sample should not exceed the determination of absolute
the value of 0.10%.
(E) calculation of the proportion of content. aluminium atoms to zirconium atoms, and content
calculation of the relative content of aluminium plus zirconium atoms to chlorine atoms content
1. calculation of ratio of aluminium atoms to content the content of zirconium atoms
Al: Zr ratio using the formula:
Al% (m/m) x 91.22
Al: Zr ratio =-------------------
ZR% (m/m) x 26.98
2. Calculation of the relative content of aluminium plus zirconium atoms to chlorine atoms content
The ratio of (Al + Zr): Cl is calculated according to the formula:
Al% (m/m) Zr% (m/m)
---------- + ----------
91.22 26.98
the ratio of (Al + Zr): Cl =--------------------------
CL% (m/m)
----------
35.45
35. The qualitative and quantitative DETERMINATION of HEXAMIDINE,
DIBROMOHEXAMIDINE, DIBROMOPROPAMIDINE AND CHLORHEXIDINE 1.
1. scope and field of application
This method describes the qualitative and quantitative determination of
-hexamidine and its salts, including the 2hydroxyethansulfonátu and the 4-
hydroxybenzoate,
-dibromohexamidine and its salts, including the 2hydroxyethansulfonátu,
-dibromopropamidine and its salts, including the 2hydroxyethansulfonátu,
chlorhexidine diacetate, digluconate and-dihydrochloride in cosmetic
resource.
2. The definition of the
Concentrations of hexamidine, bromdibromhexamidinu, bromdibrompropamidinu and
chlorhexidine determined by this method is expressed as percentage by weight
percentages (% m/m).
3. The essence of the method
Qualitative and quantitative determination is carried out by chromatography
ion pairs, using high-performance liquid chromatography (HPLC)
with the reversed with UV detection spectrophotometry.
Hexamidine, dibromhexamidin, dibromopropamidine and chlorhexidine,
qualitatively determined by their retention times in the chromatography
tailback.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher and according to
needs must be suitable for HPLC.
4.1 Methanol
4.2. Sodium-heptane-1-sulfonate monohydrate
4.3 glacial acetic acid (d20 = 1.05 g/ml)
4.4. sodium chloride
4.5 mobile phase
4.5.1
Solvent I:0,005 mol/l solution of sodium-heptane-1-sulfonate monohydrate
(4.2) in methanol (4.1), adjusted with glacial acetic acid (4.3) on the
the value of the pH 3.5.
4.5.2
Solvent II: 0.005 mol/l solution of sodium-heptane-1-sulfonate monohydrate
(4.2) in water, adjusted with glacial acetic acid (4.3) on pH value
3.5.
Note: if necessary to improve the shape of the peaks, you can edit the mobile phase and the
prepare the ways listed below:
-solvent i: 5.84 g sodium chloride (4.4) and 1.1013 g of sodium
heptane-1-sulfonate monohydrate (4.2) in 100 ml of water. Adds
with 900 ml of methanol (4.1) and adjust with glacial acetic acid (4.3)
on the value of the pH 3.5.
-solvent II: 5.84 g sodium chloride (4.4) and 1.1013 g of sodium
heptane-1-sulfonate monohydrate (4.2) in one litre of water and
adjusts with glacial acetic acid (4.3) to a pH of 3.5.
4.6 Hexamidindi (2hydroxyethansulfonát) [C20H26N4O22C2H6O4S]
4.7 Dibromhexamidindi (2hydroxyethansulfonát) [C20H24Br2N4O22C2H6O4S]
4.8 Dibrompropamidindi (2hydroxyethansulfonát) [C17H18Br2N4O22C2H6O4S]
4.9 Chlorhexidindiacetát [C22H30Cl2N102C2H4O2]
4.10 standard solutions:
in solvent I (4.5.1) prepare 0.05% (m/V) solutions of each of the four
preservatives (4.6 to 4.9).
4.11 3, 4, 4 ' Trichlorkarbanilid (Triclocarban-INN)
4.12.4,4 ' Dichlor3 (trifluormethyl) karbanilid (Halocarban-INN)
5. apparatus and equipment
5.1. Normal laboratory equipment
5.2 high-performance liquid chromatograph chromatography with UV
with a variable wavelength detector
5.3. analytical column:
stainless steel, length 30 cm, internal diameter 4 mm, with filling
mikroBondapack C18, 10 mikrom, or equivalent.
5.4. ultrasonic bath
6. Qualitative determination
6.1. Preparation of the sample
Into volumetric flasks 10 ml, weigh about 0.5 g of the sample and make up
volume with solvent I (4.5.1). The flask for 10 minutes in an ultrasonic inserts
Bath (5.4). Filter or centrifuge. The filtrate or
supernatant for chromatography.
6.2 the Chromatography
6.2.1. Mobile-phase Gradient
-------------------------------------------------------
| | | Time eluent I eluent II |
| (min) | (% V/V) (4.5.1) | (% V/V) (4.5.2) |
-------------------------------------------------------
|0 |50 |50 |
|15 |65 |35 |
|30 |65 |35 |
|45 |50 |50 |
-------------------------------------------------------
6.2.2.
The flow rate of the mobile phase (6.2.1) is set to 1.5 ml/min and column temperature
is set to 35 ° C.
6.2.3.
Detector wavelength to 264 nm sets.
6.2.4
Inject 10 mikrol each of the reference solutions (4.10) and
record their chromatograms.
6.2.5
Inject a 10 mikrol of the sample solution (6.1) and record its
the chromatogram.
6.3.
Identify whether hexamidine, dibromohexamidine, dibromopropamidine or
chlorhexidine is present by comparing the retention times of the peaks recorded
in point 6.2.5 with those obtained from the reference solutions in 6.2.4 point.
7. The quantitative determination of
7.1 preparation of standard solutions
As the internal standard shall apply one of the preservatives (4.6 to 4.9),
that is not present in the sample. It is not possible, it can be used
Triclocarban (4.11) or Halocarban (4.12).
7.1.1.
Preservative stock solution, whose presence is established in point 6.3,
0.05% (m/V) in solvent I (4.5.1).
7.1.2.
Stock solution of the preservative chosen as in internal standard 0.05% (m/V)
in solvent I (4.5.1).
7.1.3.
For each found a preservative, prepare four standard solutions
that the volumetric flasks 10 ml volumetric according to the table below
the quantity of the stock solution of proven preservative (7.1.1) and quantity
the stock solution of internal standard (7.1.2).
------------------------------------------------------------
Standard stock solution of preservative stock solution,
the internal solution whose presence was
proven standard
the number of added mikrog/ml
added ml (7.1.1) (*)
ml (7.1.2)
------------------------------------------------------------
And 1.0 0.5 25
(II) 1.0 1.0 a 50
(III) 1.0 1.5 75
(IV) 1.0 2.0 100
------------------------------------------------------------
(*) These values are given as an indication and correspond to the
concentrations of the standard solutions, proven preservatives,
prepared from stock solutions containing exactly
0.05% of the evidence of the preservative.
7.2. Preparation of the sample
7.2.1.
Into volumetric flasks 10 ml, weigh approximately 0.5 g (p g) of the sample,
Add 1.0 ml of internal standard solution (7.1.2) and 6 ml of solvent I
(4.5.1) and mix.
7.2.2.
Place the flask for 10 minutes in the ultrasonic bath (5.4). Cool.
Make up to volume with solvent I and mix. Centrifuge or filter
through a fluted filter paper. The supernatant or filtrate
apply for chromatography.
7.3 Chromatography
7.3.1.
Mobile-phase gradient, flow rate, column temperature and detector wavelength
the HPLC equipment (5.2) are set according to the conditions required in the
qualitative determination (6.2.1 to 6.2.3).
7.3.2.
Inject 10 mikrol of the sample solution (7.2.2) and measure the peak areas.
This procedure is repeated with the other 10 mikrol aliquots of solution
the sample so long as they are given identical results. It shall be calculated
the ratio of the peak area of the peak area analysis to compounds of the internal
standard.
7.4 Calibration
7.4.1
Inject 10 mikrol of each standard solution (7.1.3) and measure the
peak areas.
7.4.2.
For each standard solution (7.1.3), calculate the ratio of the area of the peak
hexamidine, dibromohexamidine, dibromopropamidine or chlorhexidine to
the peak area of the internal standard. The calibration curve by plotting the
These ratios on the y axis and the corresponding concentrations of the standard
solutions, proven preservatives in mikrog/ml on the horizontal axis.
7.4.3.
From the calibration curve (7.4.2) read off the concentration of evidence
the preservative corresponding to the peak area ratio calculated in paragraph 7.3.2.
8. The calculation of the
8.1.
The contents of the hexamidine, dibromohexamidine, dibromopropamidine or
chlorhexidine in the sample, in percentage by mass, is given by
formula:
(c) the MW1
% (m/m) = ---------- x --------
1000 x p MW2
where
p = weight of the sample taken for analysis (7.2.1) in (g),
c = concentration of the preservative in the sample solution, as recorded
from the calibration curve, in mikrog/ml,
MW1 = molecular weight of the basic form of the preservative present,
Mw2 = molecular weight of the corresponding salt (see point 10).
9. Repeatability
For products containing about 0.1% (m/m) of hexamidine, dibromohexamidine,
dibromopropamidine or chlorhexidine, the difference between the results of two
parallel on the same sample should not exceed the determination of the absolute value of the
0.005%.
10. Table of formula weights
Hexamidine C20H26N4O2 354.45
Hexamidindi (2hydroxyethansulfonát) C20H26N4O 2.2 C2H6O4S 606.72
Hexamidindiphydroxybenzoát C20H26N4O 2.2 630.71 C7H6O3
C20H24Br2N4O2 512.24 Dibromhexamidin
Dibromhexamidindi C2H6O4S 764.51 C20H24Br2N4O 2.2
(2hydroxyethansulfonát)
Dibromopropamidine C17H18Br2N4O2 470.18
Dibrompropamidindi C2H6O4S 722.43 C17H18Br2N4O 2.2
(2hydroxyethansulfonát)
Chlorhexidine C22H30Cl2N10 505.45
Chlorhexidindiacetát 625.56 C22H30Cl2N 10.2 Ch3cooh
Chlorhexidindiglukonát C22H30Cl2N 10.2 C6H12O7 897.76
Chlorhexidindihydrochlorid C22H30Cl2N 10.2 HCl 578.37
36. The qualitative and quantitative DETERMINATION of BENZOIC ACID, ACID
4-Hydroxybenzoic, sorbic acid, salicylic acid and ACID
PROPIONIC ACID IN COSMETIC PRODUCTS
1. scope and field of application
Method is applicable to the qualitative and quantitative determination of
benzoic acid, 4-Hydroxybenzoic acid, sorbic acid,
Salicylic acid and propionic acid in cosmetic products.
Separate procedures describe the qualitative determination of these
preservatives, quantitative determination of propionic acid and
quantitative determination of 4-Hydroxybenzoic acid, acid
Salicylic acid, sorbic acid and benzoic acid.
2. The definition of the
The quantity of benzoic acid, 4-Hydroxybenzoic acid, acid
Salicylic acid, sorbic acid and propionic acid determined by this
method is expressed in percentage by mass of the free acids.
AND QUALITATIVE DETERMINATION
1. The principle of the
After the acid/alkaline extraction of the preservatives, the extract analyses
thin-layer chromatography (TLC) and the reaction chromatography
("derivatisation at the Board"). Depending on the results of the qualitative
determination of high-performance confirms liquid chromatography (HPLC) or,
in the case of propionic acid, by gas chromatography (GC).
2. USED CHEMICALS
2.1 General
All the reagents must be of analytical purity or higher. Must be
use distilled water or water of at least equivalent purity.
2.2 the Acetone
2.3. diethyl ether
2.4 Acetonitrile
2.5 Toluene
2.6 nHexan
2.7. Paraffin, liquid
2.8. hydrochloric acid 4 mol/l
2.9. potassium hydroxide, 4 mol/l aqueous solution
2.10. Calcium chloride, CaCl 2.2 H2O
2.11. Lithium carbonate, Li2CO3
2.12 2Brom2 ' acetonafton
2.13 Acid 4-Hydroxybenzoic acid
2.14. Salicylic acid
2.15. Benzoic acid
2.16. Sorbic acid
2.17. Propionic acid
2.18 the standard solutions
Prepare 0.1% (m/V) solutions (100 mg/100 ml) each of the five
preservatives (2.13 to 2.17) in diethyl ether.
2.19. Derivatization reagent
0.5% solution of 2bromo2 ' acetonaftonu (2.12) in acetonitrile (2.4) (50 mg/10
ml). This solution should be prepared weekly and stored in the
the refrigerator.
2.20. catalyst Solution
0.3% (m/V) solution of lithium carbonate (2.11) in water (300 mg/100 ml).
This solution should be prepared fresh.
2.21 the mobile phase
Toluene (2.5)/acetone (2.2) (20:0.5, in/in)
2.22. Liquid paraffin (2.7),/nhexan (2.6) (1:2, V/V)
3. apparatus and equipment
Usual laboratory equipment
3.1. water bath, capable of maintaining a temperature of 60 ° C
3.2. Developing tank
3.3. Ultraviolet light source, 254 and 366 nm
3.4 the finished plates for thin-layer chromatography, silica gel 60, without
fluorescence indicator, 20 × 20 cm, layer thickness 0.25 mm with
concentrating zone 2.5 × 20 cm (Merck 11845, or equivalent)
3.5. Microsyringe, 10 mikrol
3.6. Microsyringe, 25 mikrol
3.7. Oven regulated to a temperature up to 105 ° C
3.8. conical flasks, 50 and 200 ml, with ground-glass stopper
3.9. Filter paper, diameter 90 mm, Schleicher & schull, Weissband No.
5892, or equivalent
3.10. Universal pH indicator paper, pH 1-11
3.11 the Glass sample vial to the 5 ml
3.12 rotary vacuum evaporator (Rotavapor or equivalent)
3.13 the heating plate
4. the procedure
4.1. Preparation of samples
In an Erlenmeyer flask of 50 ml, with ground-glass stopper (3.8) the dares
approximately 1 g of the sample. Add four drops of 4 mol/l acid
(2.8) and 40 ml of acetone (2.2). For strongly basic
resources, such as toilet SOAP, it should be added 20 drops 4
mol/l hydrochloric acid (2.8). Using indicator paper
(3.10), check that the pH is about 2. Stopper and for
1 minute to shake strongly.
Drove necessary to facilitate the extraction of preservatives into the acetone phase, the mixture is
carefully heated to about 60 ° C, the liquid phase.
The solution is cooled to room temperature and filter through a filter
paper (3.9) into a conical flask.
20 ml of the filtrate into a 200 ml Conical flask, add 20
ml of water and mix with the pH of the mixture to approximately 10 with 4
mol/l potassium hydroxide (2.9), using indicator paper
(3.10).
Add 1 g calcium chloride (2.10) and shake vigorously. Filter
through a filter paper (3.9) into a 250 ml separating funnel containing 75
ml diethyl ether (2.3) and shake vigorously for 1 minute. The layers are
to separate, and the aqueous layer into a 250 ml Erlenmeyer flask.
The ether layer is destroyed. Using indicator paper (3.10)
the pH of an aqueous solution to approximately 2 4 mol/l
hydrochloric acid (2.8). Add 10 ml of diethyl ether (2.3),
stopper the flask and shake vigorously for 1 minute; the layers are
to separate and transfer the ether layer to a rotating film evaporator (3.12). The aqueous
the layer is destroyed.
Evaporate the ether layer almost to dryness and redissolve the residue in 1
ml diethyl ether (2.3). Transfer the solution into a sample vial
(3.11).
4.2. Thin-layer chromatography
On the start line in the concentration zone of a TLC plate (3.4) into points,
the number of which corresponds to the number of the reference solutions and the samples
to be chromatographed, apply a syringe (3.5) in the same
intervals after 3 mikrol of lithium carbonate (2.20) and dry in
a stream of cold air.
Transfer the TLC plate on the heating plate (3.13) heated to 40
St. (C) to make the spots as small as possible. Microsyringe (3.5)
the starting line of the plate apply 10 mikrol each of the standard
solution (2.18) and of the sample solution (4.1) to the exact spots where the form was
Lithium carbonate.
Finally, again on the exact spots where the reference have been deposited
solutions/sample solutions and solution of lithium carbonate, spotted approximately
15 mikrol derivatising reagent (2.19) (2bromo2 ' acetonaftonu).
Heat the TLC plate in an oven (3.7) 45 minutes at 80th.
(C). When cool, the Board develops in the Chamber (3.2), which was 15 minutes
(without unloading filter paper) sycena mobile phase 2.21
(toluene/acetone), until the solvent front has reached a distance of 15 cm
(the time of the development is approximately 80 minutes).
The plate is dried in a stream of cold air and gained the spots
evaluated under UV light (3.3). To enhance the fluorescence of the weak spots
TLC plate may be submerged in liquid paraffin/n-hexane
(2.22).
5. Qualitative determination
For each spot is calculated the value of the Rf.
The Rf values and behavior pattern under UV light compared to the values and
the behavior of the reference samples.
Take the preliminary conclusions about the presence of and qualitative determination
preservatives. Perform the HPLC described
in section B, or ukazujeli, that is the presence of propionic acid,
gas chromatography is performed as described in section C of the retention.
times are compared with those for the reference samples.
The results of the TLC and HPLC or GC are combined and the ultimate proof of preservatives
based on the combination of results.
(B). The QUANTITATIVE DETERMINATION of BENZOIC ACID, acid-4-
HYDROXYBENZOIC ACID, SORBIC ACID AND SALICYLIC ACID
1. The principle of the
After acidification of the sample extracted with a mixture of ethanol and water. After filtration, the
preservatives provides high-performance liquid chromatography (HPLC).
2. USED CHEMICALS
2.1
All the reagents must be of analytical purity or higher and must be
suitable for HPLC. Used must be distilled water or water of at least
equivalent purity.
2.2. Ethanol, absolute
2.3 acid, 4-Hydroxybenzoic acid
2.4. Salicylic acid
2.5. Benzoic acid
2.6. Sorbic acid
2.7. sodium acetate (CH3COONa. 3H2O)
2.8. acetic acid, d204 = 1.05 g/ml
2.9 Acetonitrile
2.10. sulfuric acid 2 mol/l
2.11. potassium hydroxide, 0.2 mol/l aqueous solution
2.12 2-methoxybenzoic acid
2.13. ethanol/water Mixture
Nine volumes of ethanol (2.2) is mixed with one part water (2:1)
2.14. Internal standard solution
Prepare a solution containing 1 g 2-methoxybenzoic acid (2.12)
500 ml ethanol/water mixture (2.13).
2.15. Mobile phase for HPLC
2.15.1
Acetate buffer: to 1 l of water add 6.35 g acetate
sodium (2.7) and 20.0 ml acetic acid (2.8) and mix.
2.15.2
The mobile phase by mixing nine volumes acetate prepare parts tlumivýho
solution (2.15.1) and one part of Acetonitrile (2.9).
2.16. preservative stock solution
Into volumetric flasks, 50 ml, weigh about 0.05 g of 4-
hydroxybenzoic acid (2.3), 0.2 g salicylic acid (2.4), 0.2 g of
benzoic acid (2.5) and 0.05 g sorbic acid (2.6) and volume
ethanol/water mixture (2.13). Keep the solution in a refrigerator. The solution is
stable for one week.
2.17. Standard preservative solutions
To 20 ml volumetric flasks, add 8.00; 4.00; 2.00; 1.00 and 0.50 ml
stock solution (2.16). To each flask, add 10.00 ml solution
the internal standard (2.14) and 0.5 ml of 2 mol/l sulphuric acid (2.10).
The volume with ethanol/water mixture (2.13). These solutions must be
prepared fresh.
3. apparatus and equipment
Usual laboratory equipment, which is not specified and
3.1. water bath, set at 60 ° C
3.2 Chromatograph for high-performance liquid chromatography with UV
with a variable wavelength detector and 10-µl injection mikrol
loop
3.3. Analytical column
Stainless steel, length 12.5 to 25 cm, internal diameter 4.6 mm, filled with
Nucleosilem 5C18 or equivalent
3.4. Filter paper, diameter 90 mm, Schleicher and schull, Weissband No
5892, or equivalent
3.5. conical flasks, 50 ml
3.6 Glass sample vial to the 5 ml
3.7. Boiling chips, carborundum, size 2 to 4 mm, or equivalent
4. the procedure
4.1. Preparation of the sample
4.1.1. Sample preparation without addition of internal standard
In a 50-ml Erlenmeyer flask (3.5) weigh 1 g of the sample. Into the flask
transfer by pipette 1.00 ml of 2 mol/l sulphuric acid (2.10) and 40.0 ml of the mixture
ethanol/water mixture (2.13). Add approximately 1 g of boiling chips (3.7),
close the tube and shake vigorously for at least 1 minute, until the
homogeneous suspension is obtained. In order to facilitate the extraction of the preservatives into the
the ethanol phase flask for exactly 5 minutes in a water bath (3.1)
at 60 ° C.
Immediately cool the flask in a stream of cold water and store the extract at the hour
at 5 ° C.
Filter the extract through a filter paper (3.4). About 2 ml of the extract
converted into a sample vial (3.6). The extract is kept at 5 ° C
and the HPLC determination within 24 hours.
4.1.2 sample preparation with the addition of internal standard
In a 50-ml Erlenmeyer flask (3.5) to the nearest three decimal
space weigh 1 +/-0.1 g of the sample (or grams). Add by pipette 1.00 ml 2
mol/l sulphuric acid (2.10) and 30.0 ml ethanol/water mixture (2.13). Service item
approximately 1 g of boiling chips (3.7) and 10.00 ml of the internal
standard. Stopper and shake vigorously for at least 1 minute
until a homogeneous suspension is obtained. In order to facilitate the extraction of the preservatives
into the ethanol phase heat exactly 5 minutes in a water bath (3.1)
at 60 ° C.
Immediately cool the tube in a stream of cold water and store the extract is placed on
hour at 5 ° C.
Filter the extract through a filter paper (3.4). About 2 ml of the extract
converted into a sample vial (3.6). The extract is kept at 5 ° C
and the HPLC determination within 24 hours.
4.2 high performance liquid chromatography
Mobile phase: acetonitrile/acetate buffer (2.15).
The flow rate of the mobile phase through the column to 2.0 +/-0.5 ml/min.
Detector wavelength to 240 nm.
4.2.1 Calibration
Into the liquid chromatograph (3.2), inject 10 mikrol
standard solutions of preservatives (2.17). For each of the solution
obtained determine the ratio of the height of the peak investigated
the preservative to the peak height of the internal standard peak obtained from the chromatograms.
Plot the graph of the dependence on the ratio of the peak heights of the concentration of each
the standard solution.
Make sure that the calibration was obtained for standard solutions
linear response.
4.2.2 Determination
Into the liquid chromatograph (3.2), inject 10 mikrol extract
sample (4.1.1) and record the chromatogram. Inject 10 mikrol
standard preservative solution (2.17) and record the chromatogram.
Received chromatograms. If in the chromatogram of the extract
sample (4.1.1) no peak having approximately the same retention time as peak
2-methoxybenzoic acid (recommended internal standard), inject
10 into the liquid chromatograph mikrol sample extract with added
the internal standard (4.1.2) and record the chromatogram.
They rode in the chromatogram of the sample extract (4.1.1) interfering peak is the same
retention time as 2-methoxybenzoic acid, another should be selected
the internal standard. (If one of the investigated preservatives in
the chromatogram of the present, this can be used as a preservative to the internal
standard.)
Make sure that the calibration was for standard solutions
acquired linear response.
It is necessary to verify whether the chromatograms obtained for a standard solution and
the sample solution meet the following requirements:
-resolution of the peaks of the worst separated pair shall be at least 0.90. (Definition
the resolution of the peaks is shown in Figure 12);
(f)
p = ---
(g)
Figure 12: the resolution of the peaks
If the desired resolution is achieved, it should be either used
more efficient column or should be the composition of the mobile phase adjusted
that request was fulfilled;
-the asymmetry factor As of all peaks obtained shall range between 0.9
to 1.5. (Definition of the asymmetry factor is shown on Figure 13) When
record the chromatogram for the determination of the asymmetry factor is
the recommended speed of the paper 2 cm/min.
(b)
As =-
and
Figure 13: peak asymmetry factor
-Base must be stable.
5. The calculation of the
For the calculation of the concentration of the preservatives in the sample solution apply ratios
the peak heights of the investigated preservatives to the height of the peak 2-
methoxybenzoové (internal standard) and the calibration graph. The contents of the
benzoic acid, 4-Hydroxybenzoic acid, sorbic acid or
Salicylic acid in the sample, in percentage by mass (xi), using
using the formula
100. 20. (b) (b)
xi % (m/m) = ------------- = --------
106. and 500. and
where
a = mass of the test sample (4.1.2) (g),
b = the concentration of the preservative in the sample extract (4.1.2)
(mikrol/ml) obtained from the calibration graph.
6. Repeatability
For products containing about 0.4% (m/m) of 4-Hydroxybenzoic acid must not
the difference between the results of two parallel determinations on the same sample
shall not exceed an absolute value of 0.035%.
For products containing about 0.5% (m/m) the difference between the benzoic acid
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.050%.
For products containing about 0.5% (m/m) of salicylic acid, the difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.045%.
For products containing about 0.6% (m/m) the difference between the sorbic acid
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.035%.
7. Notes
7.1
The results of testing the robustness of the method shows that the quantity of acid
sulphuric acid added to the extract of the sample is critical, and the amount of
sample should be within the specified range.
7.2.
If desired, you can use an appropriate protective column.
(C). THE QUANTITATIVE DETERMINATION OF PROPIONIC ACID
1. scope and field of application
This method is suitable for the determination of propionic acid in cosmetic products
resources up to a maximum concentration of 2% (m/m).
2. The definition of the
The concentration of propionic acid determined by this method is expressed in
percentage by mass (% m/m) of the resource.
3. The principle of the
After the extraction of propionic acid from a resource with the quantitative determination
perform gas chromatography using 2-methylpropionic acid
as the internal standard.
4. CHEMICALS USED
All the reagents must be of analytical purity or higher and must be
use distilled water or water of at least equivalent purity.
4.1. Ethanol, 96% (V/V)
4.2. Propionic acid
acid 2methylpropionová
4.4. Orthophosphoric acid, 10% (m/V)
4.5. propionic acid solution
Into a 50 ml volumetric flask, weigh 1.00 g (p gram) of acid
propionic acid and make up to volume with ethanol (4.1).
4.6. Internal standard solution
Into a 50 ml volumetric flask, weigh, exactly 1.00 g (e grams) of 2-
methylpropionové and make up to volume with ethanol (4.1).
5. apparatus and equipment
5.1. Normal laboratory equipment
5.2. Gas Chromatograph, with flame ionization detector
5.3 Glass tube (20 × 150 mm) with screw cap
5.4. water bath, set at 60 ° C
5.5 Glass syringe with filter membrane 10 ml (diameter
pore mikrom 0.45)
6. the procedure
6.1. Preparation of the sample
6.1.1. Sample preparation without the internal standard
Into a glass tube (5.3), weigh about 1 g of the sample accurately. Add
0.5 ml of phosphoric acid (4.4) and 9.5 ml of ethanol (4.1).
Close the tube and shake vigorously. If necessary, place the
place the tube for five minutes in a water bath heated to 60 ° C (5.4), in order to
completely dissolve the lipid phase. Place the cooled quickly under running
water. Part of the solution is filtered through a membrane filter (5.5).
Chromatography of filtrate is carried out the same day.
6.1.2. Sample preparation with the internal standard
Into a glass tube (5.3) with a precision of three decimal places weigh 1
+/-0.1 g of the sample. Add 0.5 ml of phosphoric acid (4.4),
0.50 ml of internal standard solution and 9 ml ethanol (4.1).
Close the tube and shake vigorously. If necessary, place the
place the tube for five minutes in a water bath heated to 60 ° C (5.4), in order to
completely dissolve the lipid phase. Place the cooled quickly under running
water. Part of the solution is filtered through a membrane filter (5.5).
Chromatography of filtrate is carried out the same day.
6.2 Conditions for gas chromatography
The following are recommended operating conditions
Column
Type of stainless steel
Length 2 m
Diameter 3 mm
Filling 10% SPTM 1000 (or equivalent) + 1% H3PO4 on chromosorb WAW
100-120 mesh
The temperature of the
Injector 200 ° C
Column 120 ° C
Detector 200 ° C
Carrier gas nitrogen
Flow rate 25 ml/min
6.3 Chromatography
6.3.1 Calibration
Into a series of 20-ml volumetric flasks transfer by pipette 0.25; 0.50; 1.00; 2.00 and
4.00 ml propionic acid solution (4.5). To each volumetric flask
transfer by pipette 1.00 ml internal standard solution (4.6); make up to
volume with ethanol and mix. Thus the solutions prepared contain acid
2-methylpropionic as internal standard at a concentration of e mg/ml (it
1 mg/ml if e = 1.000) and propionic acid in a concentration
p/4 p/2, p, 2 p, 4 p mg/ml (0.25; 0.50; 1.00; 2.00 4.00;
mg/ml If p = 1,000).
Inject 1 mikrol these solutions and calibration curve by
plotting the ratio of the propionic acid 2-
methylpropionové on the x-axis and the ratio of the corresponding peak areas on the y axis.
Inject 3 benefits of each solution and calculates the average of the ratios
areas of the peak.
6.3.2 quantitative determination
Inject 1 mikrol of the sample filtrate 6.1.1. The chromatogram is compared with the
chromatogramem one of the standard solutions (6.3.1). If peak
approximately the same retention time as 2-methylpropionic acid peak,
changes to the internal standard. It is not observed any interference,
inject 1 mikrol of the sample filtrate 6.1.2 and measure the peak area
propionic acid peak and the internal standard.
Inject 3 benefits of each solution and calculates the average of the ratios
areas of the peak.
7. Calculations
7.1
From the calibration curve constructed under 6.3.1 subtracts the ratio
weight (to), which corresponds to the peak area ratio calculated in point
6.3.2.
7.2.
From the ratio of mass thus obtained calculate the propionic acid content
in the sample (x) in percentage by mass using the formula:
0.5. 100. (e) (e)
x % (m/m) = K -------------- = K ---
50 and
where
K = the ratio calculated in point 4.4,
e = the mass, in g of the internal standard as specified in section 4.6,
a = weight of sample in g referred to in section 6.1.2.
The results are rounded to one decimal place.
8. Repeatability
For products containing about 2% (m/m) of propionic acid, the difference
between the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.12%.
37. The qualitative and quantitative DETERMINATION of HYDROQUINONE, HYDROQUINONE-
MONOMETHYLETHERU, HYDROQUINONE AND HYDROQUINONE-MONOETHYLETHERU-
MONOBENZYLETHERU IN COSMETIC PRODUCTS
AND QUALITATIVE DETERMINATION
1. scope and field of application
Method describes the detection and quantitative determination of HYDROQUINONE,
Hydroquinone-hydroquinone-monomethyletheru, monoethyletheru and
Hydroquinone-monobenzyletheru (monobenzone) in cosmetic products
for lightening the skin.
2. The essence of the method
Qualitative determination of hydroquinone and its ethers shall be
thin-layer chromatography (TLC).
3. CHEMICALS USED
All the reagents must be of analytical purity or higher
3.1. Ethanol, 96% (V/V)
3.2. Chloroform
3.3. diethyl ether
3.4. mobile phase: Chloroform/diethyl ether, 66:33 (in/in)
3.5. Ammonia, 25% (m/m) (d204 = 0.91 g/ml)
3.6. Ascorbic acid
3.7 Hydroquinone
3.8. Hydroquinone monomethylether-
3.9. Hydroquinone monoethylether-
3.10. Hydroquinone monobenzylether (monobenzon)-
3.11. Standard solutions
The following standard solutions are prepared fresh and are stable
one day.
3.11.1.
In a 10-ml graduated test tube weigh 0.05 g hydroquinone (3.7). Adds
is 0.250 g of Ascorbic acid (3.6) and 5 ml of ethanol (3.1). Add
ammonia (3.5) until the pH is 10 and make up on ethanol
Volume 10 ml (3.1).
3.11.2
In a 10-ml graduated test tube weigh 0.05 g hydroquinone-
monomethyletheru (3.8). Add 0.250 g of Ascorbic acid (3.6) and 5
ml ethanol (3.1). Add ammonia (3.5) until the pH is 10,
and make up to a volume of 10 ml with ethanol (3.1).
3.11.3
In a 10-ml graduated test tube weigh 0.05 g hydroquinone-
monoethyletheru (3.9). Add 0.250 g of Ascorbic acid (3.6) and 5 ml
ethanol (3.1). Add ammonia (3.5) until the pH is 10 and
make up to a volume of 10 ml with ethanol (3.1).
3.11.4
In a 10-ml graduated test tube weigh 0.05 g hydroquinone-
monobenzyletheru (3.10). Add 0.250 g of Ascorbic acid (3.6) and 5
ml ethanol (3.1). Add ammonia (3.5) until the pH is 10,
and make up to a volume of 10 ml with ethanol (3.1).
3.12. silver nitrate
3.13 12molybdofosforečná Acid
3.14. potassium Ferricyanide hexahydrate
3.15. Ferric Chloride, hexahydrate
3.16 the detection reagents
3.16.1
Up to 5% aqueous solution (m/V) of silver nitrate (3.12), add
ammonia (3.5) until the precipitate that forms dissolves.
Warning:
When the longer standing in solution form explosive compounds and should be
the use of the discarded.
3.16.2.
10% (m/V) solution of 12molybdofosforečné acid (3.13) in ethanol (3.1).
3.16.3
Prepare a 1% (m/V) aqueous solution of potassium ferricyanide (3.14) and
2% (m/V) aqueous solution of ferric chloride (3.15). Just before use
Mix equal parts of both solutions.
4. apparatus and equipment
Normal laboratory equipment, and
4.1 Normal equipment for thin-layer chromatography
4.2. TLC plates Ready: silica gel GHR/UV254;; 20 x 20 cm (Machery, Nagel
or equivalent). Layer thickness 0.25 mm.
4.3. ultrasonic bath
4.4 Centrifuge
4.5 UV lamp, 254 nm
5. the procedure
5.1. Preparation of the sample
In a 10-ml graduated test tube weigh 3.0 g of the sample. Add 0.250 g
Ascorbic acid (3.6) and 5 ml of ethanol (3.1). the pH of the solution is adjusted
ammonia (2.2) to 10. Make up the volume with ethanol (3.1) in 10 ml.
Close the tube with the stopper and homogenize for 10 minutes in an ultrasonic
the bath. Filter through a filter paper or centrifuge at 3 000
RPM.
5.2. Thin-layer chromatography (TLC)
5.2.1.
Saturate with the mobile phase (3.4).
5.2.2.
Deposit on a plate 2 mikrol standard solutions (3.11) and 2 mikrol
the sample solution (5.1). Develops in the dark at ambient temperature until the forehead
the solvent reaches a distance of 15 cm from the start.
5.2.3.
The plate, allow to dry at room temperature.
5.3 Detection
5.3.1.
The plate under UV light at 254 nm, and the position of the spots.
5.3.2.
Spray the plate with
-silver nitrate (3.16.1), or
-12molybdofosforečnou acid (3.16.2); heat to approximately 120 ° C;
or
-potassium ferricyanide solution and ferric chloride solution
(3.16.3).
6. Qualitative determination
Calculate the Rf value of each of the spots.
The spots obtained for the sample solution shall compare the spots obtained for the
standard solutions, in terms of their Rf values, the color of the stain in the UV
the light and color of the spots after visualization detection reagent.
Perform the HPLC described in
the following section (B), and compare the retention times obtained for peak
the sample (s) with those peak times the reference solutions.
The results from TLC and HPLC combined with the aim to prove the presence of the
hydroquinone and/or its ethers.
7. Notes
Under these conditions, the following Rf values were observed:
Hydroquinone: 0.32
Hydroquinone monomethylether: 0.53-
Hydroquinone monoethylether: 0.55-
Hydroquinone monobenzylether: 0.58-
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
The method specifies a procedure for the determination of HYDROQUINONE,
Hydroquinone-hydroquinone-monomethyletheru, monoethyletheru and
Hydroquinone-monobenzyletheru in cosmetic products for lightening
skin.
2. The principle of the
The sample is extracted with a mixture of water/methanol mixture under gentle heating setting
dissolve grease material. Quantitative determination of analytes in the resulting
the solution is carried out with reversed phase liquid chromatography with UV
detection.
3. CHEMICALS USED
3.1
All the reagents must be of analytical purity or higher. Must be
use distilled water or water of at least equivalent purity.
3.2. Methanol
3.3 Hydroquinone
3.4. Hydroquinone monomethylether-
3.5. Hydroquinone monoethylether-
3.6. Hydroquinone monobenzylether (monobenzon)-
3.7. Tetrahydrofuran, HPLC grade
3.8 a mixture of water/methanol mixture 1:1 (V/V).
Mix one part water and one part of methanol (3.2).
3.9. mobile phase: tetrahydrofuran/water mixture 45:55 (V/V). Mix 45
volumes of tetrahydrofuran (3.7) and 55 parts water.
3.10 standard solution
Into a 50 ml volumetric flask, weigh 0.06 g hydroquinone (3.3), 0.08 g
Hydroquinone-monomethyletheru (3.4), 0.10 g hydroquinone-monoethyletheru
(3.5) and 0.12 g hydroquinone-monobenzyletheru (3.6). Dissolve and make up to
to the volume with methanol (3.2). The standard solution is prepared by diluting the
10.00 ml of this solution to 50.00 ml with water/methanol mixture (3.8). These
solutions should be freshly prepared.
4. apparatus and equipment
Usual laboratory equipment
4.1 water bath capable of maintaining a temperature of 60 ° C
4.2.
High-performance liquid chromatograph chromatography with UV
with a variable wavelength detector and 10-µl injection mikrol
loop
4.3. Analytical column:
Stainless steel chromatographic column, length 250 mm, internal diameter
4.6 mm, filled with Zorbax fenylem (chemically bound fenethylsilanem on
Zorbax SIL, closed trimethylchlorsilanem), particle size 6 mikrom,
or equivalent. Not used, with the exception of guard column
phenyl columns or columns with equivalent.
4.4.
Filter paper, diameter 90 mm, Schleicher and schull, Weissband No 5892
or equivalent
5. the procedure
5.1. Preparation of the sample
Into volumetric flasks, 50 ml, to the nearest three decimal places weigh
1 +/-0.1 g (a grams) of the sample. The sample is dispersed in 25 ml of a mixture of
water/methanol mixture (3.8). Stopper and shake vigorously until the
homogeneous suspension is obtained. Shake for at least 1 minute. The flask
placed in the water bath (4.1), set at 60 ° C, in order to facilitate the
the extraction. Cool the flask and make up to volume with water/methanol
(3.8) of the filter the extract through a filter paper (4.4). Qualitative
HPLC determination within 24 hours of preparation of the extract.
5.2 high performance liquid chromatography
5.2.1.
The flow rate of the mobile phase (3.9) to 1.0 ml/min and the wave
the length of the detector is set to 295 nm.
5.2.2.
Inject 10 mikrol of the sample solution obtained in 5.1 and
record the chromatogram. Measure the peak areas. The calibration is performed
According to section 5.2.3. Compare the chromatograms obtained for a sample and
standard solutions. To calculate the concentration of the analyte in the sample solution
apply peak areas and the response factors (RF) calculated under 5.2.3.
5.2.3 Calibration
Inject 10 mikrol of the standard solution (3.10) and record
the chromatogram. Inject a few times until the peak area
constant.
Determine the response factor RFi:
PI
RFI =-----
CI
where
PI = peak area for HYDROQUINONE, hydroquinone monomethylether,-
Hydroquinone monoethylether or hydroquinone--monobenzylether
CI = concentration of HYDROQUINONE, hydroquinone-monomethyletheru,
Hydroquinone-hydroquinone-monoethyletheru or
monobenzyletheru in the standard solution (3.10) (50 g/ml).
It is necessary to verify whether the chromatograms obtained for a standard solution and
the sample solution meet the following requirements:
-resolution of the peaks of the worst separated pair shall be at least 0.90. (Definition
the peak resolution is given in Figure 14);
(f)
p = ---
(g)
Picture of the l4: resolution of the peaks
If the desired resolution is achieved, it should be either used
more efficient column or should be the composition of the mobile phase adjusted
that request was fulfilled;
-the asymmetry factor As of all peaks obtained shall range between 0.9
to 1.5. (Definition of the asymmetry factor is listed in Figure 15) When
record the chromatogram for the determination of the asymmetry factor is
recommended recording speed of 2 cm/min.
Figure 15: peak asymmetry factor
-Base must be stable.
6. The calculation of the
To calculate the concentration of the analyte (s) in the sample is
apply the peak areas of the analytes. The concentration of the analyte in the sample, expressed in
percentage by mass (xi) shall be calculated using the formula
BI. 100
xi % (m/m) = ----------
RFI. and
where
a = weight of the sample in grams,
BI = peak area of the analyte in the sample.
7. Repeatability
7.1
For products containing about 2% (m/m) of HYDROQUINONE, the difference between
the results of two parallel determinations on the same sample must not exceed
the absolute value of 0.13%.
7.2.
For products that contain about 1% (m/m) of hydroquinone-monomethyletheru must not
the difference between the results of two parallel determinations on the same sample
shall not exceed an absolute value of 0,1%.
7.3.
For products that contain about 1% (m/m) of hydroquinone-monoethyletheru must not
the difference between the results of two parallel determinations on the same sample
shall not exceed an absolute value of 0,11%.
7.4.
For products that contain about 1% (m/m) of hydroquinone-monobenzyletheru must not
the difference between the results of two parallel determinations on the same sample
shall not exceed an absolute value of 0,11%.
8. The reproducibility of the
8.1.
For products containing about 2% (m/m) of hydroquinone must not absolute
the value of the difference between the results of two parallel determinations on the same
a sample made by the same method under different conditions (different
laboratories, different operators, different apparatus and/or different time),
shall not exceed an absolute value of 0.37%.
8.2.
For products that contain about 1% (m/m) of hydroquinone-monomethyletheru must not
the absolute value of the difference between the results of two parallel determinations on
the same sample, by the same method carried out under different conditions (different
laboratories, different operators, different apparatus and/or different time),
shall not exceed an absolute value of 0.21%.
8.3
For products that contain about 1% (m/m) of hydroquinone-monoethyletheru must not
the absolute value of the difference between the results of two parallel determinations on
the same sample under different conditions (different laboratories, different
operators, different apparatus and/or different time), may not exceed
the absolute value of 0,19%.
5.2
For products that contain about 1% (m/m) of hydroquinone-monobenzyletheru must not
the absolute value of the difference between the results of two parallel determinations on
the same sample, by the same method carried out under different conditions (different
laboratories, different operators, different apparatus and/or different time),
shall not exceed an absolute value of 0,11%.
9. Notes
9.1.
Rode a hydroquinone content considerably higher than 2% and is required
an accurate estimate of the content, the sample extract (5.1) dilute to a similar
concentration, you should sample containing 2% hydroquinone, and
Repeat the determination.
(In some instruments can when high concentrations of hydroquinone
lie outside of the absorbance of the linear range of the detector.)
5.7 interference
The above described method allows the determination of hydroquinone and its ethers in
one izokratickém over. The use of phenyl column shall ensure
sufficient retention for HYDROQUINONE, which cannot be guaranteed when using
C18 columns with the mobile phase.
In this method, however, easily leads to the rušivému of the influence of a wide range of parabens.
In this case, the determination should be repeated with another system
the mobile phase/stationary phase. The appropriate method can be found in the links (7)
(8):
Column: Zorbax ODS, 4.6 mm × 25 mm, or equivalent:
temperature: 36 ° C
flow: 1.5 ml/min
Mobile phase:
for HYDROQUINONE: methanol/water 5/95 (V/V)
for HYDROQUINONE monomethylether:-methanol/water 30/70 (V/V)
for HYDROQUINONE monobenzylether:-methanol/water 80/20 (V/V), ^ 8).
Column: Spherisorb S5ODS or equivalent:
Mobile phase: water/methanol 90/10 (V/V)
flow: 1.5 ml/min
These conditions are suitable for HYDROQUINONE ^ 9).
38. The qualitative and QUANTITATIVE DETERMINATION of 2FENOXYETHANOLU, 1-a
FENOXYPROPAN2OLU, METHYL, ETHYL, PROPYL, BUTYL And BENZYL4HYDROXYBENZOÁTU
IN COSMETIC PRODUCTS
AND QUALITATIVE DETERMINATION
1. scope and field of application
This method specifies a procedure, thin-layer chromatography
that, in combination with the method of quantitative determination, as described in
section (B), allows the identification of the 2fenoxyethanolu, 1fenoxypropan2olu,
methyl4hydroxybenzoátu, propyl4hydroxybenzoátu, ethyl 4-hydroxybenzoate,
butyl4hydroxybenzoátu and benzyl4hydroxybenzoátu in cosmetic
resource.
2. The principle of the
The preservatives are extracted from the okyselených cosmetic samples
resources with acetone. After filtration, the acetone solution is mixed with water and
fatty acids in an alkaline environment precipitated as calcium salts.
Alkaline acetone/water mixture is extracted with diethyl ether, which is
Removes the Lipophilic substances. After acidification the preservatives are extracted
with diethyl ether. An aliquot of the diethylether extract is spotted on the
a plate covered with a thin layer of silica gel. After development is obtained
Chromatogram of the watches in the UV light and visualized using Millon's
reagents.
3. CHEMICALS USED
3. l Generally
All the reagents must be of analytical purity or higher must be
use distilled water or water of at least the same purity.
3.2 Acetone
3.3. diethyl ether
3.4 nPentan
3.5. Methanol
3.6. acetic acid, glacial
3.7. hydrochloric acid solution, 4 mol/l
3.8. potassium hydroxide solution, 4 mol/l
3.9. Calcium chloride dihydrate (CaCl 2.2 H2O)
3.10. detection reagent: Millon's reagent
Millon's reagent (mercuric nitrate) is a commercially available solution
(Fluka 69820).
3.11 2Fenoxyethanol
3.12 1Fenoxypropan2ol
3.13 Methyl4hydroxybenzoát (methylparaben)
3.14 Ethyl4hydroxybenzoát (ethylparaben)
3.15 nPropyl4hydroxybenzoát (propylparaben)
3.16 nButyl4hydroxybenzoát (butyl)
3.17 Benzyl4hydroxybenzoát (benzylparaben)
3.18 the standard solutions
Prepare 0.1% (m/V) solutions of each of the reference substances 3.11, 3.12,
3.13, 3.14, 3.15, 3.16 and 3.17 in methanol.
3.19 the mobile phase
Mix 88 parts npentanu (3.4) with 12 parts of glacial acetic acid
(3.6).
4 apparatus and equipment
Normal laboratory equipment, and
4.1 water bath capable of maintaining a temperature of 60 ° C
4.2. Developing tank (not lined with filter paper)
4.3. Ultraviolet light source, 254 nm
4.4 the finished plates, 20 cm × 20 cm, layer of silica gel 60F254 absorption:
(fluorescent indicator), the thickness of the layer: 0.25 mm with concentrating
25 x 200 mm zone (Merck No 11798, Darmstadt, or equivalent)
4.5. Oven regulated to a temperature up to 105 ° C
4.6 hot air dryer
4.7. Woollen paint roller, length approximately 10 cm, outside diameter
approximately 3.5 cm. Woolly layer should be 2 to 3 mm thick. According to the
appropriate, the wave of cuts.
See note under 5.2.
4.8 the Erlenmeyer flask to 50 and 200 ml, with ground-glass stopper
4.9. Electric heater with thermostat.
Temperature setting: about 80 ° C heating plate is covered with aluminium
plate size 20 cm x 20 cm and a thickness of about 6 mm, achieving
uniform distribution of heat.
5. the procedure
5.1. Preparation of the sample
In an Erlenmeyer flask of 50 ml, with ground-glass stopper (4.8) weigh
approximately 1 g of the sample. Add four drops of acid solution
(3.7) and 40 ml of acetone.
In the case of strongly basic cosmetic products, such as toilet
SOAP, add 20 drops of hydrochloric acid. The flask
closed, the mixture is gently heated to about 60 ° C, in order to facilitate the
the extraction of the preservatives into the acetone phase, and 1 minute are strongly
shake.
Indicator paper to measure the pH of the solution and modifies the value? 3
hydrochloric acid solution. Again shake for 1 minute.
Cool the solution to room temperature and filter through a filter
the paper into a conical flask. 20 ml of the filtrate into a
The 200 ml conical flask, add 60 ml of water and mix thoroughly. The value of the
the pH of the mixture to potassium hydroxide (3.8) approximately 10 per
using indicator paper.
Add 1 g calcium chloride dihydrate (3.9) and shake vigorously.
Filter the solution through a filter paper into a 250 ml separating funnels,
that contains 75 ml diethylether and shake vigorously for 1 minute.
Allow the phases to separate and the aqueous phase into a conical flask
to 200 ml. The pH of the solution is adjusted acid solution
to approximately 2 with hydrochloric, using pH indicator paper. Then
Add 10 ml diethyl ether and shake vigorously 1 minute. Phase
allow to separate and transfer approximately 2 ml of diethyl ether phase converts to a sample
The vials to 5 ml.
5.2. thin-layer Chromatografíe (TLC)
The TLC plate is placed on the heated aluminium plate (4.9.) At the starting
the line in the concentration zone of the TLC plate apply 10 mikrol each of
reference solutions and 100 mikrol solution of the sample (sample) (5.1).
You can promote the evaporation of the solvent with a stream of air. TLC
the plate is removed from the heating plate and allow to cool to room
temperature. Developing tank (4.2) with 100 ml of mobile phase (3.19).
The TLC plate immediately inserted into the unsaturated Chamber and develop at
at ambient temperature until the solvent front has advanced about 15 cm from the
the base line. The plate is removed from the tank and dry in a stream of
hot air using hot-air hair dryer.
Examine the plate under UV light (4.3) and the position of the spots. Plate
heat for 30 minutes in the oven (4.5) at 100 ° C, in order to eliminate the
excess acetic acid. The preservatives in the chromatogram are
the visibility of Millonovým reagent (3.10) so that the roller (4.7)
in the reagent TLC plate moves to evenly damp.
Note: you can also make visible Stains by gently spraying drops
Millon's reagent on each spot marked under UV light.
Esters of 4-Hydroxybenzoic acid is reflected as red spots, 2-
phenoxyethanol and 1fenoxypropan2ol as yellow spots. However, it is necessary to give
Note that the acid, 4-Hydroxybenzoic acid which may be present in the
formulas as a preservative or decomposition product of Parabens,
about yourself as a red spot. See 7.3 and 7.4.
6. Qualitative determination
For each spot is calculated by the Rf value obtained from the solution Spots.
the sample is compared with the spots obtained from the reference solutions, if
as for their Rf values, their behaviour under UV light and color after
the visibility. Draw preliminary conclusions about the identity of the preservatives.
If it seems that parabens are present, the analysis
high-performance liquid chromatography (HPLC), as described in section (B).
The results of the thin-layer chromatography (TLC) and high-performance liquid
chromatography (HPLC) are combined, in order to confirm the presence of 2-
1fenoxypropan2olu and phenoxyethanol, parabens.
7. Notes
7.1
Due to the toxicity of Millon's reagent it is best to apply these
Agent one of the described methods. Spraying is not recommended.
7.2.
Other compounds containing hydroxyl groups may also respond
with Millonovým reagent giving coloring. A table of colours and Rf-values
obtained for a number of preservatives using thin-layer chromatografíe
It is mentioned in the work of n. de Kruijf, M.A.H. Rijk, L.A. PranatoSoetardhi and
A. Schouten (1987): Determination of preservatives in cosmetic products
Also: Thinlayer chromatographic procedure for the identification of
preservatives in cosmetic products (j. Chromatography 410, 395411).
7.3.
Rf-values listed in the following table are used as indicators of values,
that can be obtained:
------------------------------------------------------
Compound hRf Color
------------------------------------------------------
Acid, 4-Hydroxybenzoic acid 11 Red
Methylparaben 12 red
Ethylparaben 17 Red
Propylparaben 21 Red
Butyl 26 Red
Benzylparaben 16 Red
2-phenoxyethanol 29 yellow
1-phenoxypropan-2-ol 50 yellow
------------------------------------------------------
7.4.
In the case of 4-Hydroxybenzoic acid and methylparaben and even in the case of
benzylparaben and ethylparaben. Qualitative
the determination of these compounds should be confirmed by the HPLC method described
in section (B), and by comparing the retention times of the sample with those times
standards.
(B). THE QUANTITATIVE DETERMINATION OF
1. scope and field of application
This method specifies a procedure for the determination of 2fenoxyethanolu, 1-a
fenoxypropan2olu, methyl4hydroxybenzoátu, ethyl 4-hydroxybenzoate,
propyl4hydroxybenzoátu, butyl4hydroxybenzoátu, and benzyl4hydroxybenzoátu
in cosmetic products.
2. The definition of the
The quantity of the preservatives determined by this method is expressed in
percentage by weight.
3. The principle of the
The sample is acidified by adding sulphuric acid and then suspended in
a mixture of ethanol and water. The mixture is gently warmed to dissolve fat
phase, to ensure quantitative extraction, and then filter.
The preservatives in the filtrate is determined by high-performance liquid
chromatography (HPLC) with inverted phases using isopropyl-
4hydroxybenzoátu as internal standard.
4. CHEMICALS USED
4.1 General
All the reagents must be of analytical purity or higher and according to
needs must be suitable for HPLC. Distilled water must be used
or water of at least the same purity.
4.2. Ethanol, absolute
4.3 2Fenoxyethanol
4.4 1Fenoxypropan2ol
4.5 Methyl4hydroxybenzoát (methylparaben)
4.6 Ethyl4hydroxybenzoát (ethylparaben)
4.7 nPropyl4hydroxybenzoát (propylparaben)
4.8 Isopropyl4hydroxybenzoát (isopropylparaben)
4.9 nButyl4hydroxybenzoát (butyl)
4.10 Benzyl4hydroxybenzoát (benzylparaben)
4.11 Tetrahydrofuran
4.12 Methanol
4.13 Acetonitrile
4.14. Sulfuric acid solution, 2 mol/l
4.15. ethanol/water Mixture
Mix nine volumes of ethanol (4.2) and one volume of water.
4.16. Internal standard solution
Weigh approximately 0.25 g isopropylparaben (4.8), transfer
in a 500 ml volumetric flask, dissolve and make up to the volume of the mixture
ethanol/water mixture (4.15).
4.17. mobile phase: tetrahydrofuran/water/methanol/acetonitrile mixture
Mix 5 volumes of tetrahydrofuran, 60 volumes of water, 10 volumes of methanol and 25
parts of Acetonitrile.
4.18. stock solution of standards
In a 100 ml volumetric flask, weigh accurately approximately 0.2 g 2-
phenoxyethanol, 0.2 g 1fenoxypropan2olu, 0.05 g methylparaben, 0.05 g
ethylparaben, 0.05 g propylparaben, 0.05 g butylparaben and 0.025 g
benzyl 4-hydroxybenzoate, dissolve and make up to volume with ethanol/water mixture.
Keep the solution in the refrigerator and is stable for 1 week.
4.19. Standard preservative solutions
The volumetric flasks, 50 ml of the stock solution (4.18)
20.00 ml, 10.00 ml, 5.00 ml, 2.00 ml and 1.00 ml. To each flask, add
10.00 ml internal standard solution (4.16) and 1.0 ml of acid solution
(4.14) and make up to volume with ethanol/water mixture. These solutions
prepared fresh.
5. apparatus and equipment
Usual laboratory equipment and
5.1. water bath, capable of maintaining a temperature of 60 ° C +/-1 ° C
5.2 the HPLC chromatograph with a UV-detector, wavelength 280 nm
5.3. analytical column:
Stainless steel, length 25 cm, internal diameter 4.6 mm, length 12.5 cm (or
and internal diameter 4.6 mm) filled with Nucleosilem 5C18 or equivalent
(see 6.3).
5.4. conical flasks, 100 ml, with ground-glass stopper
5.5. Boiling chips, carborundum, size 2 to 4 mm, or equivalent
6. the procedure
6. l sample preparation
6.1.1. Sample preparation without addition of internal standard
To Erlenmeyerov 100 ml flask with ground-glass stopper, weigh approximately 1.0
(g) of the sample accurately. Pipette into the flask 1.0 ml of acid solution
(4.14) and 50.0 ml ethanol/water mixture (4.15). Add approximately 1
g of boiling chips (5.5), stopper and shake vigorously until
homogeneous suspension is obtained. Shake for at least 1 minute. The flask
Inserts for 5 minutes in a water bath (5.1) kept at 60 ° C +/-1st. C,
to support the extraction of the preservatives into the ethanol phase.
The flask immediately cool under a stream of cold water and store the extract is placed on
one hour in the refrigerator. Filter the extract through a filter paper.
Approximately 2 ml of the filtrate into a 5-ml sample vial. Extracts
stored in the refrigerator and perform the HPLC determination within 24 hours.
6.1.2. Sample preparation including addition of internal standard
To Erlenmeyerov 100 ml flask with ground-glass stopper, to the nearest
three decimal places 1.0 g/dare +-0.1 g of the sample.
Pipette into the flask 1.0 ml sulfuric acid solution (4.14) and 40.0
ml ethanol/water mixture (4.15). Add approximately 1 g of boiling chips
(5.5) and exactly 10.00 ml internal standard solution. Stopper and
shake vigorously until a homogeneous suspension is obtained. Shake
at least 1 minute. Insert the flask for 5 minutes in a water bath (5.1)
temperature 60 ° C +/-1st. (C) to support the extraction of the preservatives into the
the ethanol phase.
The flask immediately cool under a stream of cold water and store the extract is placed on
one hour in the refrigerator. Filter the extract through a filter paper.
Approximately 2 ml of the filtrate into a 5-ml sample vial (test
solution). The extract is placed in the refrigerator and perform the HPLC determination
within 24 hours.
6.2 high performance liquid chromatography (HPLC)
6.2.1. Chromatographic conditions
-Mobile phase: tetrahydrofuran/water/methanol/acetonitrile mixture (4.17)
-Flow rate: 1.5 ml/min
-Detection wavelength: 280 nm
6.2.2 Calibration
Inject 10 mikrol each of the standard preservative solutions
(4.19). Of the chromatograms obtained shall be the ratio of the peak heights of the
standard solutions of preservatives to the peak of the internal standard. For each of the
a preservative is plotted based on the concentration of these ratios
standard solutions.
6.2.3 Determination
Inject 10 to chromatogafu mikrol of the sample solution without internal
standard (6.1.1) and record the chromatogram.
Inject 10 mikrol one of the standard preservative solutions
(4.19) and record the chromatogram. The obtained chromatograms.
If in the chromatogram of the sample extract (6.1.1), the present peak
approximately the same retention time as the retention time of the peak
isopropylparaben (recommended internal standard), continue
by injecting 10 mikrol sample solution with internal standard (6.1.2).
Chromatogram and measure the peak heights.
If, in the chromatogram of the sample solution interfering peak with a retention time
approximately the same retention time as isopropylparaben, should be
another internal standard is selected.
If it is not one of the investigated preservatives in the chromatogram of the sample
present, this can be a used as an internal standard.
Calculate the ratios of the peak heights of the investigated preservatives to the peak height of the
the internal standard.
You need to verify that the calibration curve used is linear.
It is necessary to verify whether the chromatograms obtained for a standard solution and
the sample solution meet the following requirements:
-the peak separation of the couple is the least worst of separate 0.90. (Definition
peak separation is given in Figure l6.)
peak separation (p)
p = f/g
Figure 16: peak separation
If the required separation is not achieved, it should be either used more effective
the column, or it should be the composition of the mobile phase was adjusted to
requirement is met.
-the asymmetry factor (As) of all peaks obtained shall range between 0.9
to 1.5. (Definition of the asymmetry factor is shown in the figure 17.) When
record the chromatogram for the determination of the asymmetry factor is
recommended recording speed of 2 cm/min.
Factor
asymmetry (As)
As = b/a
17: peak asymmetry factor
-Base should be stable.
7. The calculation of the
For the calculation of the concentration of the preservatives in the sample solution uses
the calibration curve (6.2.2) and the values of the ratios of the peak heights of the investigated
preservatives to the peak height of the internal standard.
Content (wi), 2fenoxyethanolu, 1fenoxypropan2olu, methyl4hydroxybenzoátu,
ethyl 4-hydroxybenzoate, propyl4hydroxybenzoátu, butyl4hydroxybenzoátu and
benzyl4hydroxybenzoátu in percentage by mass (% m/m) is calculated
using the formula:
bi
% wi (m/m) = -------
200 x and
where
BI = the concentration of preservative i in the test solution (mikrog/ml)
as recorded on the calibration curve, and
a = mass of the test sample (g).
8. Repeatability
See note in section 10.5.
9. The reproducibility of the
See note in section 10.5.
10. Notes
10.1. Stationary phase
Retention behavior of rozpouštěných substances in the HPLC determinations significantly
depends on the type, brand and history of the stationary phase. Whether a column can be
used for the separation of the investigated preservatives, can be concluded from the results
obtained for standard solutions (see notes in section 6.2.3). In addition to the
the proposed material filling of the column were found to be suitable also
materials Hypersil ODS and Zorbax ODS.
In order to achieve the desired Department can, where applicable, optimize
the composition of the mobile phase.
10.2. Detection wavelength
Testing the robustness of the described method has shown that slight change
detection wavelength can have a significant effect on the results of the determination.
This parameter must be carefully controlled during analysis.
6.4 interference
Under the conditions described in this method is eluováno many other
compounds, such as preservatives and cosmetic ingredients. Retention times
large amounts of preservatives listed in annex VI to Council directive
relating to cosmetic products are listed in n. de Kruijf,
M.A.H. Rijk, L.A. PranatoSoetardhi and a. Crouching, (1989).
Determination of preservatives in cosmetic products II.
High-performance liquid chromatographic identification (J.
Chromatography 469, 317398).
10.4.
For the protection of the analytical column an appropriate guard can be used.
10.5
The method was tested in the ring, which involved nine
laboratories. Three samples were analysed. In the following table are for
each of the three samples indicated mean value in% m/m (m)
repeatability (r) and reproducibility (R) identified for the analytes,
that have been included in the samples:
----------------------------------------------------------------------------------------------------------------------
Sample 2-Fenoxy-1-Fenoxy-Butyl Benzylparaben Methylparaben Ethylparaben Propylparaben
ethanol, propan-2-ol
----------------------------------------------------------------------------------------------------------------------
Vitamin m 1.124 0.250 0.031 0.0628 0.0906
cream
r of 0.018 0.016 0.0035 0.0280 0.0044
R 0.176 0.030 0.0034 0.0068 0.0111
----------------------------------------------------------------------------------------------------------------------
Basic m 1.196 0.266 0.076
skin cream
r 0.002 0.040 0.003
R 0.147 0.022 0.004
----------------------------------------------------------------------------------------------------------------------
Massage cream 0.806 0.180 0.148 0.152 m
r 0.067 0.034 0.013 0.015
R 0.112 0.078 0.012 0.016
----------------------------------------------------------------------------------------------------------------------
1) Is released on the basis and within the limits of the law, which allows you to
incorporate the appropriate provisions of the European communities Decree.
2) first Commission directive of 22 July. December 1980 on the approximation of the laws
the laws of the Member States relating to methods of analysis necessary for the
checking the composition of cosmetic products (80/1335/EEC).
Second Commission directive of 14 July. May 1982 on the approximation of the laws,
the laws of the Member States relating to methods of analysis necessary for the
checking the composition of cosmetic products (82/434/EEC).
Third Commission directive of 27 April. September 1983 on the approximation of the laws,
the laws of the Member States relating to methods of analysis necessary for the
checking the composition of cosmetic products (83/514/EEC).
Fourth Commission directive of 11 July. October 1985 on the approximation of the laws,
the laws of the Member States relating to methods of analysis necessary for the
checking the composition of cosmetic products (85/490/EEC).
Commission directive of 10 June 1999. February 1987 amending the first directive
80/1335/EEC on the approximation of the laws of the Member States relating
the methods of analysis necessary for checking the composition of cosmetic products
resources (87/143/EEC).
Commission directive of 4 April. April 1990 amending the second directive
82/434/EEC on the approximation of the laws of the Member States relating to the
the methods of analysis necessary for checking the composition of cosmetic products
(90/207/EEC).
Fifth Commission Directive 93/73/EEC of 9 February 1976. September 1993 on the methods of analysis
necessary for checking the composition of cosmetic products.
Sixth Commission Directive 95/32/EC of 7 September 2004. July 1995 on methods
of analysis necessary for checking the composition of cosmetic products.
Seventh Commission Directive 96/45/EC of 2 December 1996. July 1996 on methods
of analysis necessary for checking the composition of cosmetic products.
8) m. HerpolBorremans et M.O. Masse, Identification et dosage de
l ' hydroquinone et de ses éthers méthylique et benzylique dans les
produits cosmétiques pour blanchir la peau. Int. j. Cosmet. Sci. 8,203,214
(1986).
9) j. Firth and i. Rix, Determination of hydroquinone in skin toning
creams, Analyst (1986), 111, p. 129.
