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Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

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2008R0440 — EN — 04.03.2016 — 006.001


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COMMISSION REGULATION (EC) No 440/2008

of 30 May 2008

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)

(OJ L 142 31.5.2008, p. 1)

Amended by:

 

 

Official Journal

  No

page

date

►M1

COMMISSION REGULATION (EC) No 761/2009 of 23 July 2009

  L 220

1

24.8.2009

►M2

COMMISSION REGULATION (EU) No 1152/2010 of 8 December 2010

  L 324

13

9.12.2010

►M3

COMMISSION REGULATION (EU) No 640/2012 of 6 July 2012

  L 193

1

20.7.2012

►M4

COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014

  L 81

1

19.3.2014

►M5

COMMISSION REGULATION (EU) No 900/2014 of 15 July 2014

  L 247

1

21.8.2014

►M6

COMMISSION REGULATION (EU) 2016/266 of 7 December 2015

  L 54

1

1.3.2016


Corrected by:

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Corrigendum, OJ L 143, 3.6.2008, p.  55 (440/2008)




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COMMISSION REGULATION (EC) No 440/2008

of 30 May 2008

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)

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THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Regulation (EC) No 1907/2006 of 18 December 2006 of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC ( 1 ), and in particular Article 13(3) thereof,

Whereas:

(1)

Pursuant to Regulation (EC) No 1907/2006, test methods are to be adopted at Community level for the purposes of tests on substances where such tests are required to generate information on intrinsic properties of substances.

(2)

Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances ( 2 ), laid down, in Annex V, methods for the determination of the physico-chemical properties, toxicity and ecotoxicity of substances and preparations. Annex V to Directive 67/548/EEC has been deleted by Directive 2006/121/EC of the European Parliament and of the Council with effect from 1 June 2008.

(3)

The test methods contained in Annex V to Directive 67/548/EEC should be incorporated into this Regulation.

(4)

This Regulation does not exclude the use of other test methods, provided that their use is in accordance with Article 13(3) of Regulation 1907/2006.

(5)

The principles of replacement, reduction and refinement of the use of animals in procedures should be fully taken into account in the design of the test methods, in particular when appropriate validated methods become available to replace, reduce or refine animal testing.

(6)

The provisions of this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:



Article 1

The test methods to be applied for the purposes of Regulation 1907/2006/EC are set out in the Annex to this Regulation.

Article 2

The Commission shall review, where appropriate, the test methods contained in this Regulation with a view to replacing, reducing or refining testing on vertebrate animals.

Article 3

All references to Annex V to Directive 67/548/EEC shall be construed as references to this Regulation.

Article 4

This Regulation shall enter into force on the day following its publication in the Official Journal of the European Union.

It shall apply from 1 June 2008.




ANNEX

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Note:

Before using any of the following test methods to test a multi-constituent substance (MCS), a substance of unknown or variable composition, complex reaction product or biological material (UVCB), or a mixture and where its applicability for the testing of MCS, UVCB, or mixtures is not indicated in the respective test method, it should be considered whether the method is adequate for the intended regulatory purpose.

If the test method is used for the testing of a MCS, UVCB or mixture, sufficient information on its composition should be made available, as far as possible, e.g. by the chemical identity of its constituents, their quantitative occurrence, and relevant properties of the constituents.

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PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES

TABLE OF CONTENTS

A.1.

MELTING/FREEZING TEMPERATURE

A.2.

BOILING TEMPERATURE

A.3.

RELATIVE DENSITY

A.4.

VAPOUR PRESSURE

A.5.

SURFACE TENSION

A.6.

WATER SOLUBILITY

A.8.

PARTITION COEFFICIENT

A.9.

FLASH-POINT

A.10.

FLAMMABILITY (SOLIDS)

A.11.

FLAMMABILITY (GASES)

A.12.

FLAMMABILITY (CONTACT WITH WATER)

A.13.

PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

A.14.

EXPLOSIVE PROPERTIES

A.15.

AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

A.16.

RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

A.17.

OXIDISING PROPERTIES (SOLIDS)

A.18.

NUMBER — AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

A.19.

LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

A.20.

SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

A.21.

OXIDISING PROPERTIES (LIQUIDS)

A.22.

LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF FIBRES

A.23.

PARTITION COEFFICIENT (1-OCTANOL/WATER): SLOW-STIRRING METHOD

A.24.

PARTITION COEFFICIENT (N-OCTANOL/WATER), HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC) METHOD

A.1.   MELTING/FREEZING TEMPERATURE

1.   METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1.   INTRODUCTION

The methods and devices described are to be applied for the determination of the melting temperature of substances, without any restriction with respect to their degree of purity.

The selection of the method is dependent on the nature of the substance to be tested. In consequence the limiting factor will be according to, whether or not the substance can be pulverised easily, with difficulty, or not at all.

For some substances, the determination of the freezing or solidification temperature is more appropriate and the standards for these determinations have also been included in this method.

Where, due to the particular properties of the substance, none of the above parameters can be conveniently measured, a pour point may be appropriate.

1.2.   DEFINITIONS AND UNITS

The melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs at atmospheric pressure and this temperature ideally corresponds to the freezing temperature.

As the phase transition of many substances takes place over a temperature range, it is often described as the melting range.

Conversion of units (K to oC)

t = T - 273,15

t

:

Celsius temperature, degree Celsius (oC)

T

:

thermodynamic temperature, kelvin (K)

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Some calibration substances are listed in the references (4).

1.4.   PRINCIPLE OF THE TEST METHOD

The temperature (temperature range) of the phase transition from the solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test substance at atmospheric pressure the temperatures of the initial melting/freezing and the final stage of melting/freezing are determined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, methods of thermal analysis, and determination of the pour point (as developed for petroleum oils).

In certain cases, it may be convenient to measure the freezing temperature in place of the melting temperature.

1.4.1.   Capillary method

1.4.1.1.   Melting temperature devices with liquid bath

A small amount of the finely ground substance is placed in a capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 1 K/min during the actual melting. The initial and final melting temperatures are determined.

1.4.1.2.   Melting temperature devices with metal block

As described under 1.4.1.1, except that the capillary tube and the thermometer are situated in a heated metal block, and can be observed through holes in the block.

1.4.1.3.   Photocell detection

The sample in the capillary tube is heated automatically in a metal cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The optical properties of most substances change from opaque to transparent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer located in the heating chamber. This method is not suitable for some highly coloured substances.

1.4.2.   Hot stages

1.4.2.1.   Kofler hot bar

The Kofler hot bar uses two pieces of metal of different thermal conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a special temperature-reading device including a runner with a pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line between the fluid and solid phase develops. The temperature at the dividing line is read by adjusting the pointer to rest at the line.

1.4.2.2.   Melt microscope

Several microscope hot stages are in use for the determination of melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive thermocouple but sometimes mercury thermometers are used. A typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains a hole permitting the entrance of light from the illuminating mirror of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area.

The heating of the sample is regulated by a rheostat. For very precise measurements on optically anisotropic substances, polarised light may be used.

1.4.2.3.   Meniscus method

This method is specifically used for polyamides.

The temperature at which the displacement of a meniscus of silicone oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.

1.4.3.   Method to determine the freezing temperature

The sample is placed in a special test tube and placed in an apparatus for the determination of the freezing temperature. The sample is stirred gently and continuously during cooling and the temperature is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for thermometer error) is recorded as the freezing temperature.

Supercooling must be avoided by maintaining equilibrium between the solid and the liquid phases.

1.4.4.   Thermal analysis

1.4.4.1   Differential thermal analysis (DTA)

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the temperature record.

1.4.4.2   Differential scanning calorimetry (DSC)

This technique records the difference in energy inputs into a substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the heat flow record.

1.4.5.   Pour point

This method was developed for use with petroleum oils and is suitable for use with oily substances with low melting temperatures.

After preliminary heating, the sample is cooled at a specific rate and examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.

1.5.   QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the melting temperature/melting range are listed in the following table:

TABLE: APPLICABILITY OF THE METHODS



A.  Capillary methods

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Melting temperature devices with liquid bath

yes

only to a few

273 to 573 K

± 0,3 K

JIS K 0064

Melting temperature with metal block

yes

only to a few

293 to > 573 K

± 0,5 K

ISO 1218 (E)

Photocell detection

yes

several with appliance devices

253 to 573 K

± 0,5 K

 

(1)   Dependent on type of instrument and on degree of purity of the substance.



B.  Hot stages and freezing methods

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Kofler hot bar

yes

no

283 to > 573 K

± 1K

ANSI/ASTM D 3451-76

Melt microscope

yes

only to a few

273 to > 573 K

± 0,5 K

DIN 53736

Meniscus method

no

specifically for polyamides

293 to > 573 K

± 0,5 K

ISO 1218 (E)

Freezing temperature

yes

yes

223 to 573 K

± 0,5 K

e.g. BS 4695

(1)   Dependent on type of instrument and on degree of purity of the substance



C.  Thermal analysis

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Differential thermal analysis

yes

yes

173 to 1 273 K

up to 600 K ± 0,5 K up to 1 273 K ± 2,0 K

ASTM E 537-76

Differential scanning calorimetry

yes

yes

173 to 1 273 K

up to 600 K ± 0,5 K up to 1 273 K ± 2,0 K

ASTM E 537-76

(1)   Dependent on type of instrument and on degree of purity of the substance



D.  Pour point

Method of measurement

Substances which can be pulverised

Substances which are not readily pulverised

Temperature range

Estimated accuracy (1)

Existing standards

Pour point

for petroleum oils and oily substances

for petroleum oils and oily substances

223 to 323 K

± 0,3 K

ASTM D 97-66

(1)   Dependent on type of instrument and on degree of purity of the substance

1.6.   DESCRIPTION OF THE METHODS

The procedures of nearly all the test methods have been described in international and national standards (see Appendix 1).

1.6.1.   Methods with capillary tube

When subjected to a slow temperature rise, finely pulverised substances usually show the stages of melting shown in figure 1.

image

During the determination of the melting temperature, the temperatures are recorded at the beginning of the melting and at the final stage.

1.6.1.1.   Melting temperature devices with liquid bath apparatus

Figure 2 shows a type of standardised melting temperature apparatus made of glass (JIS K 0064); all specifications are in millimeters.

image

A suitable liquid should be chosen. The choice of the liquid depends upon the melting temperature to be determined, e.g. liquid paraffin for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K.

For melting temperatures above 523 K, a mixture consisting of three parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such as this is used.

Only those thermometers should be used which fulfil the requirements of the following or equivalent standards:

ASTM E 1-71, DIN 12770, JIS K 8001.

The dry substance is finely pulverised in a mortar and is put into the capillary tube, fused at one end, so that the filling level is approximately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of approximately 700 mm through a glass tube vertically onto a watch glass.

The filled capillary tube is placed in the bath so that the middle part of the mercury bulb of the thermometer touches the capillary tube at the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting temperature.

The bath liquid is heated so that the temperature rise is approximately 3 K/min. The liquid should be stirred. At about 10 K below the expected melting temperature the rate of temperature rise is adjusted to a maximum of 1 K/min.

The calculation of the melting temperature is as follows:

T = TD + 0,00016 (TD - TE) n

where:

T

=

corrected melting temperature in K

TD

=

temperature reading of thermometer D in K

TE

=

temperature reading of thermometer E in K

n

=

number of graduations of mercury thread on thermometer D at emergent stem.

1.6.1.2.   Melting temperature devices with metal block

This consists of:

 a cylindrical metal block, the upper part of which is hollow and forms a chamber (see figure 3),

 a metal plug, with two or more holes, allowing tubes to be mounted into the metal block,

 a heating system, for the metal block, provided for example by an electrical resistance enclosed in the block,

 a rheostat for regulation of power input, if electric heating is used,

 four windows of heat-resistant glass on the lateral walls of the chamber, diametrically disposed at right-angles to each other. In front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps,

 a capillary tube of heat-resistant glass closed at one end (see 1.6.1.1).

See standards mentioned in 1.6.1.1. Thermoelectrical measuring devices with comparable accuracy are also applicable.

image

1.6.1.3.   Photocell detection

Apparatus and procedure:

The apparatus consists of a metal chamber with automated heating system. Three capillary are filled accordingly to 1.6.1.1 and placed in the oven.

Several linear increases of temperature are available for calibrating the apparatus and the suitable temperature rise is electrically adjusted at a pre-selected constant and linear rate. recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.

1.6.2.   Hot stages

1.6.2.1.   Kofler hot bar

See Appendix.

1.6.2.2.   Melt microscope

See Appendix.

1.6.2.3.   Meniscus method (polyamides)

See Appendix.

The heating rate through the melting temperature should be less than 1 K/min.

1.6.3.   Methods for the determination of the freezing temperature

See Appendix.

1.6.4.   Thermal analysis

1.6.4.1.   Differential thermal analysis

See Appendix.

1.6.4.2.   Differential scanning calorimetry

See Appendix.

1.6.5.   Determination of the pour point

See Appendix.

2.   DATA

A thermometer correction is necessary in some cases.

3.   REPORTING

The test report shall, if possible, include the following information:

 method used,

 precise specification of the substance (identity and impurities) and preliminary purification step, if any,

 an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see tables) is reported as the melting temperature.

If the difference between the temperature at the beginning and at the final stage of melting is within the limits of the accuracy of the method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported.

If the substance decomposes or sublimes before the melting temperature is reached, the temperature at which the effect is observed shall be reported.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 102, Decision of the Council C(81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermodynamics, Butterworths, London 1975, vol. II, p. 803-834.

(3) R. Weissberger ed.: Technique of organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII.

(4) IUPAC, Physicochemical measurements: Catalogue of reference materials from national laboratories, Pure and applied chemistry, 1976, vol. 48, p. 505-515.

Appendix

For additional technical details, the following standards may be consulted for example.

1.   Capillary methods

1.1.   Melting temperature devices with liquid bath



ASTM E 324-69

Standard test method for relative initial and final melting points and the melting range of organic chemicals

BS 4634

Method for the determination of melting point and/or melting range

DIN 53181

Bestimmung des Schmelzintervalles von Harzen nach Kapillarverfarehn

JIS K 00-64

Testing methods for melting point of chemical products

1.2.   Melting temperature devices with metal block



DIN 53736

Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen

ISO 1218 (E)

Plastics — polyamides — determination of ‘melting point’

2.   Hot stages

2.1.   Kofler hot bar



ANSI/ASTM D 3451-76

Standard recommended practices for testing polymeric powder coatings

2.2.   Melt microscope



DIN 53736

Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen

2.3.   Meniscus method (polyamides)



ISO 1218 (E)

Plastics — polyamides — determination of ‘melting point’

ANSI/ASTM D 2133-66

Standard specification for acetal resin injection moulding and extrusion materials

NF T 51-050

Résines de polyamides. Détermination du ‘point de fusion’ méthode du ménisque

3.   Methods for the determination of the freezing temperature



BS 4633

Method for the determination of crystallising point

BS 4695

Method for Determination of Melting Point of petroleum wax (Cooling Curve)

DIN 51421

Bestimmung des Gefrierpunktes von Flugkraftstoffen, Ottokraftstoffen und Motorenbenzolen

ISO 2207

Cires de pétrole: détermination de la température de figeage

DIN 53175

Bestimmung des Erstarrungspunktes von Fettsäuren

NF T 60-114

Point de fusion des paraffines

NF T 20-051

Méthode de détermination du point de cristallisation (point de congélation)

ISO 1392

Method for the determination of the freezing point

4.   Thermal analysis

4.1.   Differential thermal analysis



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis

ASTM E 473-85

Standard definitions of terms relating to thermal analysis

ASTM E 472-86

Standard practice for reporting thermoanalytical data

DIN 51005

Thermische Analyse, Begriffe

4.2.   Differential scanning calorimetry



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis

ASTM E 473-85

Standard definitions of terms relating to thermal analysis

ASTM E 472-86

Standard practice for reporting thermoanalytical data

DIN 51005

Thermische Analyse, Begriffe

5.   Determination of the pour point



NBN 52014

Echantillonnage et analyse des produits du pétrole: Point de trouble et point d'écoulement limite — Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipunt

ASTM D 97-66

Standard test method for pour point of petroleum oils

ISO 3016

Petroleum oils — Determination of pour point

A.2.   BOILING TEMPERATURE

1.   METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1.   INTRODUCTION

The methods and devices described here can be applied to liquid and low melting substances, provided that these do not undergo chemical reaction below the boiling temperature (for example: auto-oxidation, rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances.

Emphasis is put on the methods using photocell detection and thermal analysis, because these methods allow the determination of melting as well as boiling temperatures. Moreover, measurements can be performed automatically.

The ‘dynamic method’ has the advantage that it can also be applied to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement by a manostat.

Remarks:

The influence of impurities on the determination of the boiling temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.

1.2.   DEFINITIONS AND UNITS

The normal boiling temperature is defined as the temperature at which the vapour pressure of a liquid is 101,325 kPa.

If the boiling temperature is not measured at normal atmospheric pressure, the temperature dependence of the vapour pressure can be described by the Clausius-Clapeyron equation:

image

where:

p

=

the vapour pressure of the substance in pascals

Δ Hv

=

its heat of vaporisation in J mol-1

R

=

the universal molar gas constant = 8,314 J mol-1 K-1

T

=

thermodynamic temperature in K

The boiling temperature is stated with regard to the ambient pressure during the measurement.

Conversions

Pressure (units: kPa)

100 kPa

=

1 bar = 0,1 MPa

(‘bar’ is still permissible but not recommended)

133 Pa

=

1 mm Hg = 1 Torr

(the units ‘mm Hg’ and ‘Torr’ are not permitted)

1 atm

=

standard atmosphere = 101 325 Pa

(the unit ‘atm’ is not permitted)

Temperature (units: K)

t = T - 273,15

t

:

Celsius temperature, degree Celsius (oC)

T

:

thermodynamic temperature, kelvin (K)

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Some calibration substances can be found in the methods listed in the Appendix.

1.4.   PRINCIPLE OF THE TEST METHOD

Five methods for the determination of the boiling temperature (boiling range) are based on the measurement of the boiling temperature, two others are based on thermal analysis.

1.4.1.   Determination by use of the ebulliometer

Ebulliometers were originally developed for the determination of the molecular weight by boiling temperature elevation, but they are also suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at atmospheric pressure until it is boiling.

1.4.2.   Dynamic method

This method involves the measurement of the vapour recondensation temperature by means of an appropriate thermometer in the reflux while boiling. The pressure can be varied in this method.

1.4.3.   Distillation method for boiling temperature

This method involves distillation of the liquid and measurement of the vapour recondensation temperature and determination of the amount of distillate.

1.4.4.   Method according to Siwoloboff

A sample is heated in a sample tube, which is immersed in a liquid in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.

1.4.5.   Photocell detection

Following the principle according to Siwoloboff, automatic photo-electrical measurement is made using rising bubbles.

1.4.6.   Differential thermal analysis

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the temperature record.

1.4.7.   Differential scanning calorimetry

This technique records the difference in energy inputs into a substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat flow record.

1.5.   QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the boiling temperature/boiling range are listed in table 1.



Table 1:

Comparison of the methods

Method of measurement

Estimated accuracy

Existing standard

Ebulliometer

± 1,4 K (up to 373 K) (1) (2)

± 2,5 K (up to 600 K) (1) (2)

ASTM D 1120-72 (1)

Dynamic method

± 0,5 K (up to 600 K) (2)

 

Distillation process (boiling range)

± 0,5 K (up to 600 K)

ISO/R 918, DIN 53171, BS 4591/71

According to Siwoloboff

± 2 K (up to 600 K) (2)

 

Photocell detection

± 0,3 K (up to 373 K) (2)

 

Differential thermal calorimetry

± 0,5 K (up to 600 K)

± 2,0 K (up to 1 273 K)

ASTM E 537-76

Differential scanning calorimetry

± 0,5 K (up to 600 K)

± 2,0 K (up to 1 273 K)

ASTM E 537-76

(1)   This accuracy is only valid for the simple device as for example described in ASTM D 1120-72; it can be improved with more sophisticated ebulliometer devices.

(2)   Only valid for pure substances. The use in other circumstances should be justified.

1.6.   DESCRIPTION OF THE METHODS

The procedures of some test methods have been described in international and national standards (see Appendix).

1.6.1.   Ebulliometer

See Appendix.

1.6.2.   Dynamic method

See test method A.4 for the determination of the vapour pressure.

The boiling temperature observed with an applied pressure of 101,325 kPa is recorded.

1.6.3.   Distillation process (boiling range)

See Appendix.

1.6.4.   Method according to Siwoloboff

The sample is heated in a melting temperature apparatus in a sample tube, with a diameter of approximately 5 mm (figure 1).

Figure 1 shows a type of standardised melting and boiling temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres).

image

A capillary tube (boiling capillary) which is fused about 1 cm above the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the boiling capillary is fastened either to the thermometer with a rubber band or is fixed with a support from the side (see figure 2).



Figure 2

Principle according to Siwoloboff

Figure 3

Modified principle

image

image

The bath liquid is chosen according to boiling temperature. At temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid must be stirred. At about 10 K below the expected boiling temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary.

The boiling temperature is that temperature when, on momentary cooling, the string of bubbles stops and fluid suddenly starts rising in the capillary. The corresponding thermometer reading is the boiling temperature of the substance.

In the modified principle (figure 3) the boiling temperature is determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample is sucked up. The open end of the fine capillary is closed by melting, so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the substance plug reaches the level of the surface of the bath liquid (c).

1.6.5.   Photocell detection

The sample is heated in a capillary tube inside a heated metal block.

A light beam is directed, via suitable holes in the block, through the substance onto a precisely calibrated photocell.

During the increase of the sample temperature, single air bubbles emerge from the boiling capillary. When the boiling temperature is reached the number of bubbles increases greatly. This causes a change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block.

This method is especially useful because it allows determinations below room temperature down to 253,15 K (– 20 oC) without any changes in the apparatus. The instrument merely has to be placed in a cooling bath.

1.6.6.   Thermal analysis

1.6.6.1.   Differential thermal analysis

See Appendix.

1.6.6.2.   Differential scanning calorimetry

See Appendix.

2.   DATA

At small deviations from the normal pressure (max. ± 5 kPa) the boiling temperatures are normalised to Tn by means of the following number-value equation by Sidney Young:

Tn = T + (fT × Δp)

where:

Δp

=

(101,325 - p) [note sign]

P

=

pressure measurement in kPa

fT

=

rate of change of boiling temperature with pressure in K/kPa

T

=

measured boiling temperature in K

Tn

=

boiling temperature corrected to normal pressure in K

The temperature-correction factors, fT, and equations for their approximation are included in the international and national standards mentioned above for many substances.

For example, the DIN 53171 method mentions the following rough corrections for solvents included in paints:



Table 2:

Temperature — corrections factors fT

Temperature T (K)

Correction factor fT (K/kPa)

323,15

0,26

348,15

0,28

373,15

0,31

398,15

0,33

423,15

0,35

448,15

0,37

473,15

0,39

498,15

0,41

523,15

0,4

548,15

0,45

573,15

0,47

3.   REPORTING

The test report shall, if possible, include the following information:

 method used,

 precise specification of the substance (identity and impurities) and preliminary purification step, if any,

 an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see table 1) is reported as the boiling temperature.

The measured boiling temperatures and their mean shall be stated and the pressure(s) at which the measurements were made shall be reported in kPa. The pressure should preferably be close to normal atmospheric pressure.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 103, Decision of the Council C (81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, editions. Experimental thermodynamics, Butterworths, London, 1975, vol. II.

(3) R. Weissberger edition: Technique of organic chemistry, Physical methods of organic chemistry, Third Edition, Interscience Publications, New York, 1959, vol. I, Part I, Chapter VIII.

Appendix

For additional technical details, the following standards may be consulted for example.

1.   Ebulliometer

1.1. Melting temperature devices with liquid bath



ASTM D 1120-72

Standard test method for boiling point of engine anti-freezes

2.   Distillation process (boiling range)



ISO/R 918

Test Method for Distillation (Distillation Yield and Distillation Range)

BS 4349/68

Method for determination of distillation of petroleum products

BS 4591/71

Method for the determination of distillation characteristics

DIN 53171

Losungsmittel für Anstrichstoffe, Bestimmung des Siedeverlaufes

NF T 20-608

Distillation: détermination du rendement et de l'intervalle de distillation

3.   Differential thermal analysis and differential scanning calorimetry



ASTM E 537-76

Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis

ASTM E 473-85

Standard definitions of terms relating to thermal analysis

ASTM E 472-86

Standard practice for reporting thermoanalytical data

DIN 51005

Thermische Analyse, Begriffe

A.3.   RELATIVE DENSITY

1.   METHOD

The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.1.   INTRODUCTION

The methods for determining relative density described are applicable to solid and to liquid substances, without any restriction in respect to their degree of purity. The various methods to be used are listed in table 1.

1.2.   DEFINITIONS AND UNITS

The relative density D20 4 of solids or liquids is the ratio between the mass of a volume of substance to be examined, determined at 20 oC, and the mass of the same volume of water, determined at 4 oC. The relative density has no dimension.

The density, ρ, of a substance is the quotient of the mass, m, and its volume, v.

The density, ρ, is given, in SI units, in kg/m3.

1.3.   REFERENCE SUBSTANCES (1) (3)

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4.   PRINCIPLE OF THE METHODS

Four classes of methods are used.

1.4.1.   Buoyancy methods

1.4.1.1.   Hydrometer (for liquid substances)

Sufficiently accurate and quick determinations of density may be obtained by the floating hydrometers, which allow the density of a liquid to be deduced from the depth of immersion by reading a graduated scale.

1.4.1.2.   Hydrostatic balance (for liquid and solid substances)

The difference between the weight of a test sample measured in air and in a suitable liquid (e.g. water) can be employed to determine its density.

For solids, the measured density is only representative of the particular sample employed. For the determination of density of liquids, a body of known volume, v, is weighed first in air and then in the liquid.

1.4.1.3.   Immersed body method (for liquid substances) (4)

In this method, the density of a liquid is determined from the difference between the results of weighing the liquid before and after immersing a body of known volume in the test liquid.

1.4.2.   Pycnometer methods

For solids or liquids, pycnometers of various shapes and with known volumes may be employed. The density is calculated from the difference in weight between the full and empty pycnometer and its known volume.

1.4.3.   Air comparison pycnometer (for solids)

The density of a solid in any form can be measured at room temperature with the gas comparison pycnometer. The volume of a substance is measured in air or in an inert gas in a cylinder of variable calibrated volume. For the calculation of density one mass measurement is taken after concluding the volume measurement.

1.4.4.   Oscillating densitimeter (5) (6) (7)

The density of a liquid can be measured by an oscillating densitimeter. A mechanical oscillator constructed in the form of a U-tube is vibrated at the resonance frequency of the oscillator which depends on its mass. Introducing a sample changes the resonance frequency of the oscillator. The apparatus has to be calibrated by two liquid substances of known densities. These substances should preferably be chosen such that their densities span the range to be measured.

1.5.   QUALITY CRITERIA

The applicability of the different methods used for the determination of the relative density is listed in the table.

1.6.   DESCRIPTION OF THE METHODS

The standards given as examples, which are to be consulted for additional technical details, are attached in the Appendix.

The tests have to be run at 20 oC, and at least two measurements performed.

2.   DATA

See standards.

3.   REPORTING

The test report shall, if possible, include the following information:

 method used,

 precise specification of the substance (identity and impurities) and preliminary purification step, if any.

The relative density,

image

, shall be reported as defined in 1.2, along with the physical state of the measured substance.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.



Table:

Applicability of methods

Method of measurement

Density

Maximum possible dynamic viscosity

Existing Standards

solid

liquid

1.4.1.1.  Hydrometer

 

yes

5 Pa s

ISO 387,

ISO 649-2,

NF T 20-050

1.4.1.2.  Hydrostatic balance

 

 

 

 

(a)  solids

yes

 

 

ISO 1183 (A)

(b)  liquids

 

yes

5 Pa s

ISO 901 and 758

1.4.1.3.  Immersed body method

 

yes

20 Pa s

DIN 53217

1.4.2.  Pycnometer

 

 

 

ISO 3507

(a)  solids

yes

 

 

ISO 1183(B),

NF T 20-053

(b)  liquids

 

yes

500 Pa s

ISO 758

1.4.3.  Air comparison pycnometer

yes

 

 

DIN 55990 Teil 3,

DIN 53243

1.4.4.  Oscillating densitimer

 

yes

5 Pa s

 

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 109, Decision of the Council C(81) 30 final.

(2) R. Weissberger ed., Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Chapter IV, Interscience Publ., New York, 1959, vol. I, Part 1.

(3) IUPAC, Recommended reference materials for realization of physico-chemical properties, Pure and applied chemistry, 1976, vol. 48, p. 508.

(4) Wagenbreth, H., Die Tauchkugel zur Bestimmung der Dichte von Flüssigkeiten, Technisches Messen tm, 1979, vol. II, p. 427-430.

(5) Leopold, H., Die digitale Messung von Flüssigkeiten, Elektronik, 1970, vol. 19, p. 297-302.

(6) Baumgarten, D., Füllmengenkontrolle bei vorgepackten Erzeugnissen -Verfahren zur Dichtebestimmung bei flüssigen Produkten und ihre praktische Anwendung, Die Pharmazeutische Industrie, 1975, vol. 37, p. 717-726.

(7) Riemann, J., Der Einsatz der digitalen Dichtemessung im Brauereilaboratorium, Brauwissenschaft, 1976, vol. 9, p. 253-255.

Appendix

For additional technical details, the following standards may be consulted for example.

1.   Buoyancy methods

1.1.   Hydrometer



DIN 12790, ISO 387

Hydrometer; general instructions

DIN 12791

Part I: Density hydrometers; construction, adjustment and use

Part II: Density hydrometers; standardised sizes, designation

Part III: Use and test

ISO 649-2

Laboratory glassware: Density hydrometers for general purpose

NF T 20-050

Chemical products for industrial use — Determination of density of liquids — Areometric method

DIN 12793

Laboratory glassware: range find hydrometers

1.2.   Hydrostatic balance



ISO 1183

Method A: Methods for determining the density and relative density of plastics excluding cellular plastics

NF T 20-049

Chemical products for industrial use — Determination of the density of solids other than powders and cellular products — Hydrostatic balance method

ASTM-D-792

Specific gravity and density of plastics by displacement

DIN 53479

Testing of plastics and elastomers; determination of density



ISO 901

ISO 758

DIN 51757

Testing of mineral oils and related materials; determination of density

ASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62

ASTM D 1298

Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method

BS 4714

Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method

1.3.   Immersed body method



DIN 53217

Testing of paints, varnishes and similar coating materials; determination of density; immersed body method

2.   Pycnometer methods

2.1.   For liquid substances



ISO 3507

Pycnometers

ISO 758

Liquid chemical products; determination of density at 20 oC

DIN 12797

Gay-Lussac pycnometer (for non-volatile liquids which are not too viscous)

DIN 12798

Lipkin pycnometer (for liquids with a kinematic viscosity of less than 100 10-6 m2 s-1 at 15 oC)

DIN 12800

Sprengel pycnometer (for liquids as DIN 12798)

DIN 12801

Reischauer pycnometer (for liquids with a kinematic viscosity of less than 100. 10-6 m2 s-1 at 20 oC, applicable in particular also to hydrocarbons and aqueous solutions as well as to liquids with higher vapour pressure, approximately 1 bar at 90 oC)

DIN 12806

Hubbard pycnometer (for viscous liquids of all types which do not have too high a vapour pressure, in particular also for paints, varnishes and bitumen)

DIN 12807

Bingham pycnometer (for liquids, as in DIN 12801)

DIN 12808

Jaulmes pycnometer (in particular for ethanol — water mixture)

DIN 12809

Pycnometer with ground-in thermometer and capillary side tube (for liquids which are not too viscous)

DIN 53217

Testing of paints, varnishes and similar products; determination of density by pycnometer

DIN 51757

Point 7: Testing of mineral oils and related materials; determination of density

ASTM D 297

Section 15: Rubber products — chemical analysis

ASTM D 2111

Method C: Halogenated organic compounds

BS 4699

Method for determination of specific gravity and density of petroleum products (graduated bicapillary pycnometer method)

BS 5903

Method for determination of relative density and density of petroleum products by the capillary — stoppered pycnometer method

NF T 20-053

Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method

2.2.   For solid substances



ISO 1183

Method B: Methods for determining the density and relative density of plastics excluding cellular plastics

NF T 20-053

Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method

DIN 19683

Determination of the density of soils

3.   Air comparison pycnometer



DIN 55990

Part 3: Prüfung von Anstrichstoffen und ähnlichen Beschichtungsstoffen; Pulverlack; Bestimmung der Dichte

DIN 53243

Anstrichstoffe; chlorhaltige Polymere; Prüfung

▼M1

A.4.   VAPOUR PRESSURE

1.   METHOD

This method is equivalent to OECD TG 104 (2004).

1.1.   INTRODUCTION

This revised version of method A.4(1) includes one additional method; Effusion method: isothermal thermogravimetry, designed for substances with very low pressures (down to 10–10 Pa). In the light of needs for procedures, especially in relation to obtaining vapour pressure for substances with low vapour pressure, other procedures of this method are re-evaluated with respect to other applicability ranges.

At the thermodynamic equilibrium the vapour pressure of a pure substance is a function of temperature only. The fundamental principles are described elsewhere (2)(3).

No single measurement procedure is applicable to the entire range of vapour pressures from less than 10–10 to 105 Pa. Eight methods for measuring vapour pressure are included in this method which can be applied in different vapour pressure ranges. The various methods are compared as to application and measuring range in Table 1. The methods can only be applied for compounds that do not decompose under the conditions of the test. In cases where the experimental methods cannot be applied due to technical reasons, the vapour pressure can also be estimated, and a recommended estimation method is set out in the Appendix.

1.2.   DEFINITIONS AND UNITS

The vapour pressure of a substance is defined as the saturation pressure above a solid or liquid substance.

The SI unit of pressure, which is the pascal (Pa), should be used. Other units which have been employed historically are given hereafter, together with their conversion factors:



1 Torr

=

1 mm Hg

=

1,333 × 102 Pa

1 atmosphere

=

1,013 × 105 Pa

 

 

1 bar

=

105 Pa

 

 

The SI unit of temperature is the kelvin (K). The conversion of degrees Celsius to kelvin is according to the formula:

T = t + 273,15

where, T is the kelvin or thermodynamic temperature and t is the Celsius temperature.



Table 1

Measuring method

Substances

Estimated repeatability

Estimated reproducibility

Recommended range

Solid

Liquid

Dynamic method

Low melting

Yes

up to 25 %

1 to 5 %

up to 25 %

1 to 5 %

103 Pa to 2 × 103 Pa

2 × 103 Pa to 105 Pa

Static method

Yes

Yes

5 to 10 %

5 to 10 %

10 Pa to 105 Pa

10–2 Pa to 105 Pa (1)

Isoteniscope method

Yes

Yes

5 to 10 %

5 to 10 %

102 Pa to 105 Pa

Effusion method: vapour pressure balance

Yes

Yes

5 to 20 %

up to 50 %

10–3 to 1 Pa

Effusion method: Knudsen cell

Yes

Yes

10 to 30 %

10–10 to 1 P

Effusion method: isothermal thermogravimetry

Yes

Yes

5 to 30 %

up to 50 %

10–10 to 1 Pa

Gas saturation method

Yes

Yes

10 to 30 %

up to 50 %

10–10 to 103 Pa

Spinning rotor method

Yes

Yes

10 to 20 %

10–4 to 0,5 Pa

(1)   When using a capacitance manometer

1.3.   PRINCIPLE OF THE TEST

In general, the vapour pressure is determined at various temperatures. In a limited temperature range, the logarithm of the vapour pressure of a pure substance is a linear function of the inverse of the thermodynamic temperature according to the simplified Clapeyron-Clausius equation:

image

where:

p

=

the vapour pressure in pascals

ΔHv

=

the heat of vaporisation in J mol–1

R

=

the universal gas constant, 8,314 J mol–1 K–1

T

=

the temperature in K

1.4.   REFERENCE SUBSTANCES

Reference substances do not need to be employed. They serve primarily to check the performance of a method from time to time as well as to allow comparison between results of different methods.

1.5.   DESCRIPTION OF THE METHOD

1.5.1.   Dynamic method (Cottrell’s method)

1.5.1.1.   Principle

The vapour pressure is determined by measuring the boiling temperature of the substance at various specified pressures between roughly 103 and 105 Pa. This method is also recommended for the determination of the boiling temperature. For that purpose it is useful up to 600 K. The boiling temperatures of liquids are approximately 0,1 °C higher at a depth of 3 to 4 cm than at the surface because of the hydrostatic pressure of the column of liquid. In Cottrell’s method (4) the thermometer is placed in the vapour above the surface of the liquid and the boiling liquid is made to pump itself continuously over the bulb of the thermometer. A thin layer of liquid which is in equilibrium with vapour at atmospheric pressure covers the bulb. The thermometer thus reads the true boiling point, without errors due to superheating or hydrostatic pressure. The pump originally employed by Cottrell is shown in figure 1. Tube A contains the boiling liquid. A platinum wire B sealed into the bottom facilitates uniform boiling. The side tube C leads to a condenser, and the sheath D prevents the cold condensate from reaching the thermometer E. When the liquid in A is boiling, bubbles and liquid trapped by the funnel are poured via the two arms of the pump F over the bulb of the thermometer.



Figure 1image

Figure 2image

Cottrell pump (4)

A: Thermocouple

B: Vacuum buffer volume

C: Pressure gauge

D: Vacuum

E: Measuring point

F: Heating element c.a. 150 W

1.5.1.2.   Apparatus

A very accurate apparatus, employing the Cottrell principle, is shown in figure 2. It consists of a tube with a boiling section in the lower part, a cooler in the middle part, and an outlet and flange in the upper part. The Cottrell pump is placed in the boiling section which is heated by means of an electrical cartridge. The temperature is measured by a jacketed thermocouple, or resistance thermometer inserting through the flange at the top. The outlet is connected to the pressure regulation system. The latter consists of a vacuum pump, a buffer volume, a manostat for admitting nitrogen for pressure regulation and manometer.

1.5.1.3.   Procedure

The substance is placed in the boiling section. Problems may be encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. The apparatus is sealed at the flange and the substance degassed. Frothing substances cannot be measured using this method.

The lowest desired pressure is then set and the heating is switched on. At the same time, the temperature sensor is connected to a recorder.

Equilibrium is reached when a constant boiling temperature is recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of low melting solids, care should be taken to prevent the condenser from blocking.

After recording this equilibrium point, a higher pressure is set. The process is continued in this manner until 105 Pa has been reached (approximately 5 to 10 measuring points in all). As a check, equilibrium points must be repeated at decreasing pressures.

1.5.2.   Static method

1.5.2.1.   Principle

In the static method (5), the vapour pressure at thermodynamic equilibrium is determined at a specified temperature. This method is suitable for substances and multicomponent liquids and solids in the range from 10–1 to 105 Pa and, provided care is taken, also in the range 1 to 10 Pa.

1.5.2.2.   Apparatus

The equipment consists of a constant-temperature bath (precision of ± 0,2 K), a container for the sample connected to a vacuum line, a manometer and a system to regulate the pressure. The sample chamber (figure 3a) is connected to the vacuum line via a valve and a differential manometer (U-tube containing a suitable manometer fluid) which serves as zero indicator. Mercury, silicones and phthalates are suitable for use in the differential manometer, depending on the pressure range and the chemical behaviour of the test substance. However, based on environmental concerns, the use of mercury should be avoided, if possible. The test substance must not dissolve noticeably in, or react with, the U-tube fluid. A pressure gauge can be used instead of a U-tube (figure 3b). For the manometer, mercury can be used in the range from normal pressure down to 102 Pa, while silicone fluids and phthalates are suitable for use below 102 Pa down to 10 Pa. There are other pressure gauges which can be used below 102 Pa and heatable membrane capacity manometers can even be used at below 10–1 Pa. The temperature is measured on the outside wall of the vessel containing the sample or in the vessel itself.

1.5.2.3.   Procedure

Using the apparatus as described in figure 3a, fill the U-tube with the chosen liquid, which must be degassed at an elevated temperature before readings are taken. The test substance is placed in the apparatus and degassed at reduced temperature. In the case of a multiple-component sample, the temperature should be low enough to ensure that the composition of the material is not altered. Equilibrium can be established more quickly by stirring. The sample can be cooled with liquid nitrogen or dry ice, but care should be taken to avoid condensation of air or pump-fluid. With the valve over the sample vessel open, suction is applied for several minutes to remove the air. If necessary, the degassing operation is repeated several times.



Figure 3aimage

Figure 3bimage

When the sample is heated with the valve closed, the vapour pressure increases. This alters the equilibrium of the fluid in the U-tube. To compensate for this, nitrogen or air is admitted to the apparatus until the differential pressure indicator is at zero again. The pressure required for this can be read off the manometer or off an instrument of higher precision. This pressure corresponds to the vapour pressure of the substance at the temperature of the measurement. Using the apparatus described in figure 3b, the vapour pressure is read off directly.

The vapour pressure is determined at suitably small temperature intervals (approximately 5 to 10 measuring points in all) up to the desired temperature maximum.

Low-temperature readings must be repeated as a check. If the values obtained from the repeated readings do not coincide with the curve obtained for increasing temperature, this may be due to one of the following situations:

(i) the sample still contains air (e.g. in the case of highly viscous materials) or low-boiling substances which is or are released during heating;

(ii) the substance undergoes a chemical reaction in the temperature range investigated (e.g. decomposition, polymerisation).

1.5.3.   Isoteniscope Method

1.5.3.1.   Principle

The isoteniscope (6) is based on the principle of the static method. The method involves placing a sample in a bulb maintained at constant temperature and connected to a manometer and a vacuum pump. Impurities more volatile than the substance are removed by degassing at reduced pressure. The vapour pressure of the sample at selected temperatures is balanced by a known pressure of inert gas. The isoteniscope was developed to measure the vapour pressure of certain liquid hydrocarbons but it is appropriate for the investigation of solids as well. The method is usually not suitable for multicomponent systems. Results are subject to only slight errors for samples containing non-volatile impurities. The recommended range is 102 to 105 Pa.

1.5.3.2.   Apparatus

An example of a measuring device is shown in figure 4. A complete description can be found in ASTM D 2879-86 (6).

1.5.3.3.   Procedure

In the case of liquids, the substance itself serves as the fluid in the differential manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then filled by nitrogen. The evacuation and purge of the system is repeated twice to remove residual oxygen. The filled isoteniscope is placed in a horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer. The pressure of the system is reduced to 133 Pa and the sample is gently warmed until it just boils (removal of dissolved gases). The isoteniscope is then placed so that the sample returns to the bulb and fills the short leg of the manometer. The pressure is maintained at 133 Pa. The drawn-out tip of the sample bulb is heated with a small flame until the sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer, creating a vapour-filled, nitrogen-free space. The isoteniscope is then placed in a constant temperature bath, and the pressure of the nitrogen is adjusted until it equals that of the sample. At the equilibrium, the pressure of the nitrogen equals the vapour pressure of the substance.

image

In the case of solids, and depending on the pressure and temperature ranges, manometer liquids such as silicon fluids or phthalates are used. The degassed manometer liquid is put in a bulge provided on the long arm of the isoteniscope. Then the solid to be investigated is placed in the sample bulb and is degassed at an elevated temperature. After that, the isoteniscope is inclined so that the manometer liquid can flow into the U-tube.

1.5.4.   Effusion method: vapour pressure balance (7)

1.5.4.1.   Principle

A sample of the test substance is heated in a small furnace and placed in an evacuated bell jar. The furnace is covered by a lid which carries small holes of known diameters. The vapour of the substance, escaping through one of the holes, is directed onto a balance pan of a highly sensitive balance which is also enclosed in the evacuated bell jar. In some designs the balance pan is surrounded by a refrigeration box, providing heat dissipation to the outside by thermal conduction, and is cooled by radiation so that the escaping vapour condenses on it. The momentum of the vapour jet acts as a force on the balance. The vapour pressure can be derived in two ways: directly from the force on the balance pan and also from the evaporation rate using the Hertz-Knudsen equation (2):

image

where:

G

=

evaporation rate (kg s–1 m–2)

M

=

molar mass (g mol–1)

T

=

temperature (K)

R

=

universal gas constant (J mol–1 K–1)

P

=

vapour pressure (Pa)

The recommended range is 10–3 to 1 Pa.

1.5.4.2.   Apparatus

The general principle of the apparatus is illustrated in figure 5.

image



A:

Base plate

F:

Refrigeration box and cooling bar

B:

Moving coil instrument

G:

Evaporator furnace

C:

Bell jar

H:

Dewar flask with liquid nitrogen

D:

Balance with scale pan

I:

Measurement of temperature of sample

E:

Vacuum measuring device

J:

Test Substance

1.5.5.   Effusion method: Knudsen cell

1.5.5.1.   Principle

The method is based on the estimation of the mass of test substance flowing out per unit of time of a Knudsen cell (8) in the form of vapour, through a micro-orifice under ultra-vacuum conditions. The mass of effused vapour can be obtained either by determining the loss of mass of the cell or by condensing the vapour at low temperature and determining the amount of volatilised substance using chromatography. The vapour pressure is calculated by applying the Hertz-Knudsen relation (see section 1.5.4.1) with correction factors that depend on parameters of the apparatus (9). The recommended range is 10–10 to 1 Pa (10)(11)(12)(13)(14).

1.5.5.2.   Apparatus

The general principle of the apparatus is illustrated in figure 6.

image



1:

Connection to vacuum

7:

Threaded lid

2:

Wells from platinum resistance thermometer or temperature measurement and control

8:

Butterfly nuts

3:

Lid for vacuum tank

9:

Bolts

4:

O-ring

10:

Stainless steel effusion cells

5:

Aluminum vacuum tank

11:

Heater cartridge

6:

Device for installing and removing the effusion cells

 

 

1.5.6.   Effusion method: isothermal thermogravimetry

1.5.6.1.   Principle

The method is based on the determination of accelerated evaporation rates for the test substance at elevated temperatures and ambient pressure using thermogravimetry (10)(15)(16)(17)(18)(19)(20). The evaporation rates vT result from exposing the selected compound to a slowly flowing inert gas atmosphere, and monitoring the weight loss at defined isothermal temperatures T in Kelvin over appropriate periods of time. The vapour pressures pT are calculated from the vT values by using the linear relationship between the logarithm of the vapour pressure and the logarithm of the evaporation rate. If necessary, an extrapolation to temperatures of 20 and 25 °C can be made by regression analysis of log pT vs. 1/T. This method is suitable for substances with vapour pressures as low as 10–10 Pa (10–12 mbar) and with purity as close as possible to 100 % to avoid the misinterpretation of measured weight losses.

1.5.6.2.   Apparatus

The general principle of the experimental set-up is shown in figure 7.

image

The sample carrier plate, hanging on a microbalance in a temperature controlled chamber, is swept by a stream of dry nitrogen gas which carries the vaporised molecules of the test substance away. After leaving the chamber, the gas stream is purified by a sorption unit.

1.5.6.3.   Procedure

The test substance is applied to the surface of a roughened glass plate as a homogeneous layer. In the case of solids, the plate is wetted uniformly by a solution of the substance in a suitable solvent and dried in an inert atmosphere. For the measurement, the coated plate is hung into the thermogravimetric analyser and subsequently its weight loss is measured continuously as a function of time.

The evaporation rate vT at a definite temperature is calculated from the weight loss Δm of the sample plate by

image

where F is the surface area of the coated test substances, normally the surface area of the sample plate, and t is the time for weight loss Δm.

The vapour pressure pT is calculated on the basis of its function of evaporation rate vT:

Log pT = C + D · log vT

where C and D are constants specific for the experimental arrangement used, depending on the diameter of the measurement chamber and on the gas flow rate. These constants must be determined once by measuring a set of compounds with known vapour pressure and regressing log pT vs. log vT (11)(21)(22).

The relationship between the vapour pressure pT and the temperature T in Kelvin is given by

Log pT = A + B · 1/T

where A and B are constants obtained by regressing log pT vs. 1/T. With this equation, the vapour pressure can be calculated for any other temperature by extrapolation.

1.5.7.   Gas saturation method (23)

1.5.7.1.   Principle

Inert gas is passed, at room temperature and at a known flow rate, through or over a sample of the test substance, slowly enough to ensure saturation. Achieving saturation in the gas phase is of critical importance. The transported substance is trapped, generally using a sorbent, and its amount is determined. As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like gas chromatography, may be used to determine quantitatively the amount of material transported. The vapour pressure is calculated on the assumption that the ideal gas law is obeyed and that the total pressure of a mixture of gases is equal to the sum of the pressures of the component gases. The partial pressure of the test substance, i.e. the vapour pressure, is calculated from the known total gas volume and from the weight of the material transported.

The gas saturation procedure is applicable to solid or liquid substances. It can be used for vapour pressures down to 10–10 Pa (10)(11)(12)(13)(14). The method is most reliable for vapour pressures below 103 Pa. Above 103 Pa, the vapour pressures are generally overestimated, probably due to aerosol formation. Since the vapour pressure measurements are made at room temperature, the need to extrapolate data from high temperatures is not necessary and high temperature extrapolation, which can often cause serious errors, is avoided.

1.5.7.2.   Apparatus

The procedure requires the use of a constant-temperature box. The sketch in figure 8 shows a box containing three solid and three liquid sample holders, which allow for the triplicate analysis of either a solid or a liquid sample. The temperature is controlled to ± 0,5 °C or better.

image

In general, nitrogen is used as an inert carrier gas but, occasionally, another gas may be required (24). The carrier gas must be dry. The gas stream is split into 6 streams, controlled by needle valves (approximately 0,79 mm orifice), and flows into the box via 3,8 mm i.d. copper tubing. After temperature equilibration, the gas flows through the sample and the sorbent trap and exists from the box.

Solid samples are loaded into 5 mm i.d. glass tubing between glass wool plugs (see Figure 9). Figure 10 shows a liquid sample holder and sorbent system. The most reproducible method for measuring the vapour pressure of liquids is to coat the liquid on glass beads or on an inert sorbent such as silica, and to pack the holder with these beads. As an alternative, the carrier gas may be made to pass a coarse frit and bubble through a column of the liquid test substance.



Figure 9image

Figure 10image

The sorbent system contains a front and a backup sorbent section. At very low vapour pressures, only small amounts are retained by the sorbent and the adsorption on the glass wool and the glass tubing between the sample and the sorbent may be a serious problem.

Traps cooled with solid CO2 are another efficient way for collecting the vaporised material. They do not cause any back pressure on the saturator column and it is also easy to quantitatively remove the trapped material.

1.5.7.3.   Procedure

The flow rate of the effluent carrier gas is measured at room temperature. The flow rate is checked frequently during the experiment to assure that there is an accurate value for the total volume of carrier gas. Continuous monitoring with a mass flow-meter is preferred. Saturation of the gas phase may require considerable contact time and hence quite low gas flow rates (25).

At the end of the experiment, both the front and backup sorbent sections are analysed separately. The compound on each section is desorbed by adding a solvent. The resulting solutions are analysed quantitatively to determine the weight desorbed from each section. The choice of the analytical method (also the choice of sorbent and desorbing solvent) is dictated by the nature of the test material. The desorption efficiency is determined by injecting a known amount of sample onto the sorbent, desorbing it and analysing the amount recovered. It is important to check the desorption efficiency at or near the concentration of the sample under the test conditions.

To assure that the carrier gas is saturated with the test substance, three different gas flow rates are used. If the calculated vapour pressure shows no dependence on flow rate, the gas is assumed to be saturated.

The vapour pressure is calculated through the equation:

image

where:

p

=

vapour pressure (Pa)

W

=

mass of evaporated test substance (g)

V

=

volume of saturated gas (m3)

R

=

universal gas constant 8,314 (J mol–1 K–1)

T

=

temperature (K)

M

=

molar mass of test substance (g mol–1)

Measured volumes must be corrected for pressure and temperature differences between the flow meter and the saturator.

1.5.8.   Spinning rotor

1.5.8.1.   Principle

This method uses a spinning rotor viscosity gauge, in which the measuring element is a small steel ball which, suspended in a magnetic field, is made to spin by rotating fields (26)(27)(28). Pick-up coils allow its spinning rate to be measured. When the ball has reached a given rotational speed, usually about 400 revolutions per second, energising is stopped and deceleration, due to gas friction, takes place. The drop of rotational speed is measured as a function of time. The vapour pressure is deduced from the pressure-dependent slow-down of the steel ball. The recommended range is 10–4 to 0,5 Pa.

1.5.8.2.   Apparatus

A schematic drawing of the experimental set-up is shown in figure 11. The measuring head is placed in a constant-temperature enclosure, regulated within 0,1 °C. The sample container is placed in a separate enclosure, also regulated within 0,1 °C. All other parts of the set-up are kept at a higher temperature to prevent condensation. The whole apparatus is connected to a high-vacuum system.

image

2.   DATA AND REPORTING

2.1.   DATA

The vapour pressure from any of the preceding methods should be determined for at least two temperatures. Three or more are preferred in the range from 0 to 50 °C, in order to check the linearity of the vapour pressure curve. In case of Effusion method (Knudsen cell and isothermal thermogravimetry) and Gas saturation method, 120 to 150 °C is recommended for the measuring temperature range instead of 0 to 50 °C.

2.2.   TEST REPORT

The test report must include the following information:

 method used,

 precise specification of the substance (identity and impurities) and preliminary purification step, if any,

 at least two vapour pressure and temperature values — and preferably three or more — required in the range from 0 to 50 °C (or 120 to 150 °C),

 at least one of the temperatures should be at or below 25 °C, if technically possible according to the chosen method,

 all original data,

 a log p versus 1/T curve,

 an estimate of the vapour pressure at 20 or 25 °C.

If a transition (change of state, decomposition) is observed, the following information should be noted:

 nature of the change,

 temperature at which the change occurs at atmospheric pressure,

 vapour pressure at 10 and 20 °C below the transition temperature and 10 and 20 °C above this temperature (unless the transition is from solid to gas).

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

3.   LITERATURE

(1)  Official Journal of the European Communities L 383 A, 26-47 (1992).

(2) Ambrose, D. (1975). Experimental Thermodynamics, Vol. II, Le Neindre, B., and Vodar, B., Eds., Butterworths, London.

(3) Weissberger R., ed. (1959). Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Vol. I, Part I. Chapter IX, Interscience Publ., New York.

(4) Glasstone, S. (1946). Textbook of Physical Chemistry, 2nd ed., Van Nostrand Company, New York.

(5) NF T 20-048 AFNOR (September 1985). Chemical products for industrial use — Determination of vapour pressure of solids and liquids within a range from 10–1 to 105 Pa — Static method.

(6) ASTM D 2879-86, Standard test method for vapour pressure — temperature relationship and initial decomposition temperature of liquids by isoteniscope.

(7) NF T 20-047 AFNOR (September 1985). Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10–3 to 1 Pa — Vapour pressure balance method.

(8) Knudsen, M. (1909). Ann. Phys. Lpz., 29, 1979; (1911), 34, 593.

(9) Ambrose, D., Lawrenson, I.J., Sprake, C.H.S. (1975). J. Chem. Thermodynamics 7, 1173.

(10) Schmuckler, M.E., Barefoot, A.C., Kleier, D.A., Cobranchi, D.P. (2000), Vapor pressures of sulfonylurea herbicides; Pest Management Science 56, 521-532.

(11) Tomlin, C.D.S. (ed.), The Pesticide Manual, Twelfth Edition (2000).

(12) Friedrich, K., Stammbach, K., Gas chromatographic determination of small vapour pressures determination of the vapour pressures of some triazine herbicides. J. Chromatog. 16 (1964), 22-28.

(13) Grayson, B.T., Fosbraey, L.A., Pesticide Science 16 (1982), 269-278.

(14) Rordorf, B.F., Prediction of vapor pressures, boiling points and enthalpies of fusion for twenty-nine halogenated dibenzo-p-dioxins, Thermochimia Acta 112 Issue 1 (1987), 117-122.

(15) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection; Pesticide Science 4 (1973) 137-147.

(16) Gückel, W., Synnatschke, G., Ritttig, R., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection II. Application to Formulated Products; Pesticide Science 5 (1974) 393-400.

(17) Gückel, W., Kaestel, R., Lewerenz, J., Synnatschke, G., A Method for Determining the Volatility of Active Ingredients Used in Plant Protection. Part III: The Temperature Relationship between Vapour Pressure and Evaporation Rate; Pesticide Science 13 (1982) 161-168.

(18) Gückel, W., Kaestel, R., Kroehl, T., Parg, A., Methods for Determining the Vapour Pressure of Active Ingredients Used in Crop Protection. Part IV: An Improved Thermogravimetric Determination Based on Evaporation Rate; Pesticide Science 45 (1995) 27-31.

(19) Kroehl, T., Kaestel, R., Koenig, W., Ziegler, H., Koehle, H., Parg, A., Methods for Determining the Vapour Pressure of Active Ingredients Used in Crop Protection. Part V: Thermogravimetry Combined with Solid Phase MicroExtraction (SPME); Pesticide Science, 53 (1998) 300-310.

(20) Tesconi, M., Yalkowsky, S.H., A Novel Thermogravimetric Method for Estimating the Saturated Vapor Pressure of Low-Volatility Compounds; Journal of Pharmaceutical Science 87(12) (1998) 1512-20.

(21) Lide, D.R. (ed.), CRC Handbook of Chemistry and Physics, 81st ed. (2000), Vapour Pressure in the Range — 25 °C to 150 °C.

(22) Meister, R.T. (ed.), Farm Chemicals Handbook, Vol. 88 (2002).

(23) 40 CFR, 796. (1993). pp 148-153, Office of the Federal Register, Washington DC.

(24) Rordorf B.F. (1985). Thermochimica Acta 85, 435.

(25) Westcott et al. (1981). Environ. Sci. Technol. 15, 1375.

(26) Messer G., Röhl, P., Grosse G., and Jitschin W. (1987). J. Vac. Sci. Technol. (A), 5(4), 2440.

(27) Comsa G., Fremerey J.K., and Lindenau, B. (1980). J. Vac. Sci. Technol. 17(2), 642.

(28) Fremerey, J.K. (1985). J. Vac. Sci. Technol. (A), 3(3), 1715.

Appendix

Estimation method

INTRODUCTION

Estimated values of the vapour pressure can be used:

 for deciding which of the experimental methods is appropriate,

 for providing an estimate or limit value in cases where the experimental method cannot be applied due to technical reasons.

ESTIMATION METHOD

The vapour pressure of liquids and solids can be estimated by use of the modified Watson correlation (a). The only experimental data required is the normal boiling point. The method is applicable over the pressure range from 105 Pa to 10–5 Pa.

Detailed information on the method is given in ‘Handbook of Chemical Property Estimation Methods’ (b). See also OECD Environmental Monograph No.67 (c).

CALCULATION PROCEDURE

The vapour pressure is calculated as follows:

image

where:

T

=

temperature of interest

Tb

=

normal boiling point

Pvp

=

vapour pressure at temperature T

ΔHvb

=

heat of vaporisation

ΔZb

=

compressibility factor (estimated at 0,97)

m

=

empirical factor depending on the physical state at the temperature of interest

Further,

image

where, KF is an empirical factor considering the polarity of the substance. For several compound types, KF factors are listed in reference (b).

Quite often, data are available in which a boiling point at reduced pressure is given. In such a case, the vapour pressure is calculated as follows:

image

where, T1 is the boiling point at the reduced pressure P1.

REPORT

When using the estimation method, the report shall include a comprehensive documentation of the calculation.

LITERATURE

(a) Watson, K.M. (1943). Ind. Eng. Chem, 35, 398.

(b) Lyman, W.J., Reehl, W.F., Rosenblatt, D.H. (1982). Handbook of Chemical Property Estimation Methods, McGraw-Hill.

(c) OECD Environmental Monograph No.67. Application of Structure-Activity Relationships to the Estimation of Properties Important in Exposure Assessment (1993).

▼B

A.5.   SURFACE TENSION

1.   METHOD

The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.1.   INTRODUCTION

The described methods are to be applied to the measurement of the surface tension of aqueous solutions.

It is useful to have preliminary information on the water solubility, the structure, the hydrolysis properties and the critical concentration for micelles formation of the substance before performing these tests.

The following methods are applicable to most chemical substances, without any restriction in respect to their degree of purity.

The measurement of the surface tension by the ring tensiometer method is restricted to aqueous solutions with a dynamic viscosity of less than approximately 200 mPa s.

1.2.   DEFINITIONS AND UNITS

The free surface enthalpy per unit of surface area is referred to as surface tension.

The surface tension is given as:

N/m (SI unit) or

mN/m (SI sub-unit)

1 N/m = 103 dynes/cm

1 mN/m = 1 dyne/cm in the obsolete cgs system

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Reference substances which cover a wide range of surface tensions are given in references 1 and 3.

1.4.   PRINCIPLE OF THE METHODS

The methods are based on the measurement of the maximum force which is necessary to exert vertically, on a stirrup or a ring in contact with the surface of the liquid being examined placed in a measuring cup, in order to separate it from this surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed.

Substances which are soluble in water at least at a concentration of 1 mg/l are tested in aqueous solution at a single concentration.

1.5.   QUALITY CRITERIA

These methods are capable of greater precision than is likely to be required for environmental assessment.

1.6.   DESCRIPTION OF THE METHODS

A solution of the substance is prepared in distilled water. The concentration of this solution should be 90 % of the saturation solubility of the substance in water; when this concentration exceeds 1 g/l, a concentration of 1 g/l is used for testing. Substances with water solubility lower than 1 mg/l need not be tested.

1.6.1.   Plate method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.2.   Stirrup method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.3.   Ring method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.4.   OECD harmonised ring method

1.6.4.1.   Apparatus

Commercially available tensiometers are adequate for this measurement. They consist of the following elements:

 mobile sample table,

 force measuring system,

 measuring body (ring),

 measurement vessel.

1.6.4.1.1.    Mobile sample table

The mobile sample table is used as a support for the temperature-controlled measurement vessel holding the liquid to be tested. Together with the force measuring system, it is mounted on a stand.

1.6.4.1.2.    Force measuring system

The force measuring system (see figure) is located above the sample table. The error of the force measurement shall not exceed ± 10-6 N, corresponding to an error limit of ± 0,1 mg in a mass measurement. In most cases, the measuring scale of commercially available tensiometers is calibrated in mN/m so that the surface tension can be read directly in mN/m with an accuracy of 0,1 mN/m.

1.6.4.1.3.    Measuring body (ring)

The ring is usually made of a platinum-iridium wire of about 0,4 mm thickness and a mean circumference of 60 mm. The wire ring is suspended horizontally from a metal pin and a wire mounting bracket to establish the connection to the force measuring system (see figure).

(All dimensions expressed in millimetres)

image

1.6.4.1.4.    Measurement vessel

The measurement vessel holding the test solution to be measured shall be a temperature-controlled glass vessel. It shall be designed so that during the measurement the temperature of the test solution liquid and the gas phase above its surface remains constant and that the sample cannot evaporate. Cylindrical glass vessels having an inside diameter of not less than 45 mm are acceptable.

1.6.4.2.   Preparation of the apparatus

1.6.4.2.1.    Cleaning

Glass vessels shall be cleaned carefully. If necessary they shall be washed with hot chromo-sulphuric acid and subsequently with syrupy phosphoric acid (83 to 98 % by weight of H3PO4), thoroughly rinsed in tap water and finally washed with double-distilled water until a neutral reaction is obtained and subsequently dried or rinsed with part of the sample liquid to be measured.

The ring shall first be rinsed thoroughly in water to remove any substances which are soluble in water, briefly immersed in chromo-sulphuric acid, washed in double-distilled water until a neutral reaction is obtained and finally heated briefly above a methanol flame.

Note:

Contamination by substances which are not dissolved or destroyed by chromo-sulphuric acid or phosphoric acid, such as silicones, shall be removed by means of a suitable organic solvent.

1.6.4.2.2.    Calibration of the apparatus

The validation of the apparatus consists of verifying the zero point and adjusting it so that the indication of the instrument allows reliable determination in mN/m.

The apparatus shall be levelled, for instance by means of a spirit level on the tensiometer base, by adjusting the levelling screws in the base.

After mounting the ring on the apparatus and prior to immersion in the liquid, the tensiometer indication shall be adjusted to zero and the ring checked for parallelism to the liquid surface. For this purpose, the liquid surface can be used as a mirror.

The actual test calibration can be accomplished by means of either of two procedures:

(a) Using a mass: procedure using riders of known mass between 0,1 and 1,0 g placed on the ring. The calibration factor, Φa by which all the instrument readings must be multiplied, shall be determined according to equation (1).



image

 

where:

image

(mN/m)

m

=

mass of the rider (g)

g

=

gravity acceleration (981 cm s-2 at sea level)

b

=

mean circumference of the ring (cm)

σa

=

reading of the tensiometer after placing the rider on the ring (mN/m).

(b) Using water: procedure using pure water whose surface tension at, for instance, 23 oC is equal to 72,3 mN/m. This procedure is accomplished faster than the weight calibration but there is always the danger that the surface tension of the water is falsified by traces of contamination by surfactants.

The calibration factor, Φb by which all the instrument readings shall be multiplied, shall be determined in accordance with the equation (2):



image

 

where:

σo

=

value cited in the literature for the surface tension of water (mN/m)

σg

=

measured value of the surface tension of the water (mN/m) both at the same temperature.

1.6.4.3.   Preparation of samples

Aqueous solutions shall be prepared of the substances to be tested, using the required concentrations in water, and shall not contain any non-dissolved substances.

The solution must be maintained at a constant temperature (± 0,5 oC). Since the surface tension of a solution in the measurement vessel alters over a period of time, several measurements shall be made at various times and a curve plotted showing surface tension as a function of time. When no further change occurs, a state of equilibrium has been reached.

Dust and gaseous contamination by other substances interfere with the measurement. The work shall therefore be carried out under a protective cover.

1.6.5.   Test conditions

The measurement shall be made at approximately 20 oC and shall be controlled to within ± 0,5 oC.

1.6.6.   Performance of test

The solutions to be measured shall be transferred to the carefully cleaned measurement vessel, taking care to avoid foaming, and subsequently the measurement vessel shall be placed onto the table of the test apparatus. The table-top with measurement vessel shall be raised until the ring is immersed below the surface of the solution to be measured. Subsequently, the table-top shall be lowered gradually and evenly (at a rate of approximately 0,5 cm/min) to detach the ring from the surface until the maximum force has been reached. The liquid layer attached to the ring must not separate from the ring. After completing the measurements, the ring shall be immersed below the surface again and the measurements repeated until a constant surface tension value is reached. The time from transferring the solution to the measurement vessel shall be recorded for each determination. Readings shall be taken at the maximum force required to detach the ring from the liquid surface.

2.   DATA

In order to calculate the surface tension, the value read in mN/m on the apparatus shall be first multiplied by the calibration factor Φa or Φb (depending on the calibration procedure used). This will yield a value which applies only approximately and therefore requires correction.

Harkins and Jordan (4) have empirically determined correction factors for surface-tension values measured by the ring method which are dependent on ring dimensions, the density of the liquid and its surface tension.

Since it is laborious to determine the correction factor for each individual measurement from the Harkins and Jordan tables, in order to calculate the surface tension for aqueous solutions the simplified procedure of reading the corrected surface-tension values directly from the table may be used. (Interpolation shall be used for readings ranging between the tabular values.)



Table:

Correction of the measured surface tension

Only for aqueous solutions, ρ = 1 g/cm3

r

= 9,55 mm (average ring radius)

r

= 0,185 mm (ring wire radius)



Experimental Value (mN/m)

Corrected Value (mN/m)

Weight calibration (see 1.6.4.2.2(a))

Water calibration (see 1.6.4.2.2(b))

20

16,9

18,1

22

18,7

20,1

24

20,6

22,1

26

22,4

24,1

28

24,3

26,1

30

26,2

28,1

32

28,1

30,1

34

29,9

32,1

36

31,8

34,1

38

33,7

36,1

40

35,6

38,2

42

37,6

40,3

44

39,5

42,3

46

41,4

44,4

48

43,4

46,5

50

45,3

48,6

52

47,3

50,7

54

49,3

52,8

56

51,2

54,9

58

53,2

57,0

60

55,2

59,1

62

57,2

61,3

64

59,2

63,4

66

61,2

65,5

68

63,2

67,7

70

65,2

69,9

72

67,2

72,0

74

69,2

76

71,2

78

73,2

This table has been compiled on the basis of the Harkins-Jordan correction. It is similar to that in the DIN Standard (DIN 53914) for water and aqueous solutions (density ρ = 1 g/cm3 and is for a commercially available ring having the dimensions R = 9,55 mm (mean ring radius) and r = 0,185 mm (ring wire radius). The table provides corrected values for surface-tension measurements taken after calibration with weights or calibration with water.

Alternatively, without the preceding calibration, the surface tension call can be calculated according to the following formula:

image

where:

F

=

the force measured on the dynamometer at the breakpoint of the film

R

=

the radius of the ring

f

=

the correction factor (1)

3.   REPORTING

3.1.   TEST REPORT

The test report shall, if possible, include the following information:

 method used,

 type of water or solution used,

 precise specification of the substance (identity and impurities),

 measurement results: surface tension (reading) stating both the individual readings and their arithmetic mean as well as the corrected mean (taking into consideration the equipment factor and the correction table),

 concentration of the solution,

 test temperature,

 age of solution used; in particular the time between preparation and measurement of the solution,

 description of time dependence of surface tension after transferring the solution to the measurement vessel,

 all information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

3.2.   INTERPRETATION OF RESULTS

Considering that distilled water has a surface tension of 72,75 mN/m at 20 oC, substances showing a surface tension lower than 60 mN/m under the conditions of this method should be regarded as being surface-active materials.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 115, Decision of the Council C(81) 30 final.

(2) R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter XIV.

(3) Pure Appl. Chem., 1976, vol. 48, p. 511.

(4) Harkins, W.D., Jordan, H.F., J. Amer. Chem. Soc., 1930, vol. 52, p. 1751.

▼M4

A.6.   WATER SOLUBILITY

INTRODUCTION

1. This Test Method is equivalent to OECD Test Guideline (TG) 105 (1995). This Test Method is a revised version of the original TG 105 which was adopted in 1981. There is no difference of substance between the current version and that from 1981. Mainly the format has been changed. The revision was based on the EU Test Method ‘Water Solubility’ (1).

INITIAL CONSIDERATIONS

2. The water solubility of a substance can be considerably affected by the presence of impurities. This Test Method addresses the determination of the solubility in water of essentially pure substances which are stable in water and not volatile. Before determining water solubility, it is useful to have some preliminary information on the test substance, like structural formula, vapour pressure, dissociation constant and hydrolysis as a function of pH.

3. Two methods, the column elution method and the flask method which cover respectively solubilities below and above 10–2 g/l are described in this Test Method. A simple preliminary test is also described. It allows the determination of approximately the appropriate amount of sample to be used in the final test, as well as the time necessary to achieve saturation.

DEFINITIONS AND UNITS

4. The water solubility of a substance is the saturation mass concentration of the substance in water at a given temperature.

5. Water solubility is expressed in mass of solute per volume of solution. The SI unit is kg/m3 but g/l may also be used.

REFERENCE CHEMICALS

6. Reference chemicals do not need to be employed when investigating a test substance.

DESCRIPTION OF THE METHODS

Test conditions

7. The test is preferably run at 20 ± 0,5 °C. The chosen temperature should be kept constant in all relevant parts of the equipment.

Preliminary test

8. In a stepwise procedure, increasing volumes of water are added at room temperature to approximately 0,1 g of the sample (solid test substances must be pulverized) in a 10 ml glass-stoppered measuring cylinder. After each addition of an amount of water, the mixture is shaken for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the experiment is continued in a 100 ml measuring cylinder. The approximate solubility is given in Table 1 below under that volume of water in which complete dissolution of the sample occurs. When the solubility is low, a long time may be required to dissolve a test substance and at least 24 hours should be allowed. If, after 24 hours, the test substance is still not dissolved, more time (up to 96 hours) should be allowed or further dilution should be attempted to ascertain whether the column elution method or flask method should be used.



Table 1

ml of water for 0,1 g soluble

0,1

0,5

1

2

10

100

> 100

approximate solubility in g/l

> 1 000

1 000 to 200

200 to 100

100 to 50

50 to 10

10 to 1

< 1

Column elution method

Principle

9. This method is based on the elution of a test substance with water from a micro-column which is charged with an inert support material, previously coated with an excess of the test substance (2). The water solubility is given by the mass concentration of the eluate when this has reached a plateau as a function of time.

Apparatus

10. The apparatus consists of a microcolumn (Figure 1), maintained at constant temperature. It is connected either to a recirculating pump (Figure 2) or to a levelling vessel (Figure 3). The microcolumn contains an inert support held in place by a small plug of glasswool which also serves to filter out particles. Possible materials which can be employed for the support are glass beads, diatomaceous earth, or other inert materials.

11. The microcolumn shown in Figure 1 is suitable for the set-up with recirculating pump. It has a head space providing for five bed volumes (discarded at the start of the experiment) and the volume of five samples (withdrawn for analysis during the experiment). Alternatively, the size can be reduced if water can be added to the system during the experiment to replace the initial five bed volumes removed with impurities. The column is connected with tubing made of an inert material to the recirculating pump, capable of delivering approximately 25 ml/h. The recirculating pump can be, for example, a peristaltic or membrane pump. Care must be taken that no contamination and/or adsorption occur with the tube material.

12. A schematic arrangement using a levelling vessel is shown in Figure 3. In this arrangement the microcolumn is fitted with a one way stopcock. The connection to the levelling vessel consists of a ground glass joint and tubing made of an inert material. The flow rate from the levelling vessel should be approximately 25 ml/h.

image

Dimensions in mm

A. Connection for ground glass joint

B. Headspace

C. Interior 5

D. Exterior 19

E. Plug of glass wool

F. Stopcock

image

A. Atmospheric equilibration

B. Flowmeter

C. Microcolumn

D. Thermostatically controlled circulating pump

E. Recirculating pump

F. Two-way valve for sampling

image

A. Levelling vessel (e.g. 2,5 litres chemical flask)

B. Column

C. Fraction accumulator

D. Thermostat

E. Teflon tubing

F. Ground glass joint

G. Water line (between thermostat and column, inner diameter approximately 8 mm)

13. Approximately 600 mg of support material is transferred to a 50 ml round-bottom flask. A suitable amount of test substance is dissolved in a volatile solvent of analytical reagent quality and an appropriate amount of this solution is added to the support material. The solvent is completely evaporated, e.g. using a rotary evaporator, as otherwise water saturation of the support will not be achieved during the elution step because of partitioning on the surface. The loaded support material is soaked for two hours in approximately 5 ml of water and the suspension is poured into the microcolumn. Alternatively, dry loaded support material may be poured into the water-filled microcolumn and two hours are allowed for equilibrating.

14. The loading of the support material may cause problems, leading to erroneous results, e.g. when the test substance is deposited as an oil. These problems should be examined and the details reported.

Procedure using a recirculating pump

15. The flow through the column is started. It is recommended that a flow rate of approximately 25 ml/h, corresponding to 10 bed volumes per hour for the column described, be used. At least the first five bed volumes are discarded to remove water soluble impurities. Following this, the pump is allowed to run until equilibrium is established, as defined by five successive samples whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least ten bed volumes. Depending on the analytical method used, it may be preferable to establish a concentration/time curve to show that equilibrium is reached.

Procedure using a levelling vessel

16. Successive eluate fractions should be collected and analysed by the chosen method. Fractions from the middle eluate range, where the concentrations are constant within ± 30 % in at least five consecutive fractions, are used to determine the solubility.

17. Double distilled water is the preferred eluent. Deionized water with a resistivity above 10 megohms/cm and total organic carbon content below 0,01 % can also be used.

18. Under both procedures, a second run is performed at half the flow rate of the first. If the results of the two runs are in agreement, the test is satisfactory. If the measured solubility is higher with the lower flow rate, then the halving of the flow rate must continue until two successive runs give the same solubility.

19. Under both procedures, the fractions should be checked for the presence of colloidal matter by examination of the Tyndall effect. The presence of particles invalidates the test and the test should be repeated after improvement of the filtering action of the column.

20. The pH of each sample should be measured, preferably by using special indicator strips.

Flask method

Principle

21. The test substance (solids must be pulverized) is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved, the mixture is cooled and kept at the test temperature. Alternatively, and if it is assured by appropriate sampling that the saturation equilibrium is reached, the measurement can be performed directly at the test temperature. Subsequently, the mass concentration of the test substance in the aqueous solution, which must not contain any undissolved particles, is determined by a suitable analytical method (3).

Apparatus

22. The following materials are needed:

 normal laboratory glassware and instrumentation;

 a device for the agitation of solutions under controlled constant temperature;

 if required for emulsions, a centrifuge (preferably thermostated); and

 analytical equipment.

Procedure

23. The quantity of test substance necessary to saturate the desired volume of water is estimated from the preliminary test. About five times that quantity is weighed into each of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes, flasks). A volume of water, chosen in function of the analytical method and solubility range, is added to each vessel. The vessels are tightly stoppered and then agitated at 30 °C. A shaking or stirring device capable of operating at constant temperature should be used, e.g. magnetic stirring in a thermostated water bath. After one day, one of the vessels is equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the test temperature and the concentration of the test substance in the clear aqueous phase is determined by a suitable analytical method. The other two flasks are treated similarly after initial equilibration at 30 °C for two and three days respectively. If the concentrations measured in at least the two last vessels do not differ by more than 15 %, the test is satisfactory. If the results from vessels 1, 2 and 3 show a tendency of increasing values, the whole test should be repeated using longer equilibration times.

24. The test can also be performed without pre-incubation at 30 °C. In order to estimate the rate of establishment of the saturation equilibrium, samples are taken until the stirring time no longer influences the concentrations measured.

25. The pH of each sample should be measured, preferably by using special indicator strips.

Analytical determinations

26. A substance-specific method is preferred since small amounts of soluble impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration, photometry, voltametry.

DATA AND REPORTING

Data

Column elution method

27. For each run, the mean value and standard deviation from at least five consecutive samples taken from the saturation plateau should be calculated. The mean values obtained from two tests with different flows should not differ by more than 30 %.

Flask method

28. The individual results from each of the three flasks, which should not differ by more than 15 %, are averaged.

Test Report

Column elution method

29. The test report must include the following information:

 the results of the preliminary test

 chemical identity and impurities (preliminary purification step, if any)

 the concentrations, flow rates and pH for each sample

 the means and standard deviations from at least five samples from the saturation plateau of each run

 the average of at least two successive runs

 the temperature of the water during the saturation process

 the method of analysis

 the nature of the support material

 loading of the support material

 solvent used

 evidence of any chemical instability of the substance during the test

 all information relevant for the interpretation of the results, in particular with regard to impurities and physical state of the test substance.

Flask method

30. The test report must include the following information:

 the results of the preliminary test

 chemical identity and impurities (preliminary purification step, if any)

 the individual analytical determinations and the average where more than one value was determined for each flask

 the pH of each sample

 the average of the values for different flasks which were in agreement

 the test temperature

 the analytical method

 evidence of any chemical instability of the substance during the test

 all information relevant for the interpretation of the results, in particular with regard to impurities and physical state of the test substance.

LITERATURE:

(1) Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (OJ L 383, 29.12.1992, p. 113).

(2) NF T 20-045 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with low solubility — Column elution method.

(3) NF T 20-046 (AFNOR) (September 1985). Chemical products for industrial use — Determination of water solubility of solids and liquids with high solubility — Flask method.

▼B

A.8.   PARTITION COEFFICIENT

1.   METHOD

The ‘shake flask’ method described is based on the OECD Test Guideline (1).

1.1.   INTRODUCTION

It is useful to have preliminary information on structural formula, dissociation constant, water solubility, hydrolysis, n-octanol solubility and surface tension of the substance to perform this test.

Measurements should be made on ionisable substances only in their non-ionised form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least one pH unit below (free acid) or above (free base) the pK.

This test method includes two separate procedures: the shake flask method and high performance liquid chromatography (HPLC). The former is applicable when the log Pow value (see below for definitions) falls within the range - 2 to 4 and the latter within the range 0 to 6. Before carrying out either of the experimental procedures a preliminary estimate of the partition coefficient should first be obtained.

The shake-flask method applies only to essentially pure substances soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided).

The HPLC method is not applicable to strong acids and bases, metal complexes, surface-active materials or substances which react with the eluent. For these materials, a calculated value or an estimate based on individual n-octanol and water solubilities should be provided.

The HPLC method is less sensitive to the presence of impurities in the test compound than is the shake-flask method. Nevertheless, in some cases impurities can make the interpretation of the results difficult because peak assignment becomes uncertain. For mixtures which give an unresolved band, upper and lower limits of log P should be stated.

1.2.   DEFINITION AND UNITS

The partition coefficient (P) is defined as the ratio of the equilibrium concentrations (ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case n-octanol and water:

image

The partition coefficient (P) therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P).

1.3.   REFERENCE SUBSTANCES

Shake-flask method

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

HPLC method

In order to correlate the measured HPLC data of a compound with its P value, a calibration graph of log P versus chromatographic data using at least six reference points has to be established. It is for the user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a Pow above that of the test substance, and another a Pow below that of the test substance. For log P values less than 4, the calibration can be based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better accuracy, it is preferable to choose reference compounds which are structurally related to the test substance.

Extensive lists of values of log Pow for many groups of chemicals are available (2)(3). If data on the partition coefficients of structurally related compounds are not available, then a more general calibration, established with other reference compounds, may be used.

A list of recommended reference substances and their Pow values is given in Appendix 2.

1.4.   PRINCIPLE OF THE METHOD

1.4.1.   Shake-flask method

In order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined.

1.4.2.   HPLC method

HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. Chemicals injected onto such a column move along it at different rates because of the different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity, with water-soluble chemicals eluted first and oil-soluble chemicals last, in proportion to their hydrocarbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The partition coefficient is deduced from the capacity factor k, given by the expression:

image

in which, tr = retention time of the test substance, and to = average time a solvent molecule needs to pass through the column (dead-time).

Quantitative analytical methods are not required and only the determination of elution times is necessary.

1.5.   QUALITY CRITERIA

1.5.1.   Repeatability

In order to assure the accuracy of the partition coefficient, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ± 0,3 log units.

In order to increase the confidence in the measurement, duplicate determinations must be made. The values of log P derived from individual measurements should fall within a range of ± 0,1 log units.

1.5.2.   Sensitivity

The measuring range of the method is determined by the limit of detection of the analytical procedure. This should permit the assessment of values of log Pow in the range of - 2 to 4 (occasionally when conditions apply, this range may be extended to log Pow up to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre.

The HPLC method enables partition coefficients to be estimated in the log Pow range 0 to 6.

Normally, the partition coefficient of a compound can be estimated to within ± l log unit of the shake-flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can usually be achieved when correlation plots are based on structurally-related reference compounds (9).

1.5.3.   Specificity

The Nernst Partition Law applies only at constant temperature, pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results.

Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the concentration of the solution.

Because of the multiple equilibria involved, this test method should not be applied to ionisable compounds without applying a correction. The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance of this pH for the environment.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Preliminary estimate of the partition coefficient

The partition coefficient is estimated preferably by using a calculation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance ill the pure solvents (10).

1.6.2.   Shake-flask method

1.6.2.1.   Preparation

n-Octanol: the determination of the partition coefficient should be carried out with high purity analytical grade reagent.

Water: water distilled or double distilled in glass or quartz apparatus should be employed. For ionisable compounds, buffer solutions in place of water should be used if justified.

Note:

Water taken directly from an ion exchanger should not be used.

1.6.2.1.1.    Pre-saturation of the solvents

Before a partition coefficient is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker and then to let them stand long enough to allow the phases to separate and to achieve a saturation state.

1.6.2.1.2.    Preparation for the test

The entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilisation. The volume ratio and quantities of substance to be used are fixed by the following:

 the preliminary assessment of the partition coefficient (see above),

 the minimum quantity of test substance required for the analytical procedure, and

 the limitation of a maximum concentration in either phase of 0,01 mol per litre.

Three tests are carried out. In the first, the calculated volume ratio of n-octanol to water is used; in the second, this ratio is divided by two; and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2, 2:1).

1.6.2.1.3.    Test substance

A stock solution is prepared in n-octanol pre-saturated with water. The concentration of this stock solution should be precisely determined before it is employed in the determination of the partition coefficient. This solution should be stored under conditions which ensure its stability.

1.6.2.2.   Test conditions

The test temperature should be kept constant (± 1 oC) and lie in the range of 20 to 25 oC.

1.6.2.3.   Measurement procedure

1.6.2.3.1.    Establishment of the partition equilibrium

Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions.

The n-octanol phases should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to rotate the tube quickly through 180o about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes are recommended.

1.6.2.3.2.    Phase separation

When necessary, in order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature controlled centrifuge is used, the centrifuge tubes should be kept for equilibration at the test temperature for at least one hour before analysis.

1.6.2.4.   Analysis

For the determination of the partition coefficient, it is necessary to determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced.

The aqueous phase should be sampled by a procedure that minimises the risk of including traces of n-octanol: a glass syringe with a removable needle can be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. Examples of analytical methods which may be appropriate are:

 photometric methods,

 gas chromatography,

 high-performance liquid chromatography.

1.6.3.   HPLC method

1.6.3.1.   Preparation

A liquid chromatograph, fitted with a pulse-free pump and a suitable detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have the minimal percentage of polar groups (11). Commercial microparticulate reverse-phase packings or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

HPLC grade methanol and HPLC grade water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with a minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting compounds of log P 6 within an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time (and those of the reference compounds) by decreasing the polarity of the mobile phase or the column length.

Substances with very low solubility in n-octanol tend to give abnormally low log Pow values with the HPLC method; the peaks of such compounds sometimes accompany the solvent front. This is probably due to the fact that the partitioning process is too slow to reach the equilibrium in the time normally taken by an HPLC separation. Decreasing the flow rate and/or lowering the methanol/water ratio may then be effective to arrive at a reliable value.

Test and reference compounds should be soluble in the mobile phase in sufficient concentrations to allow their detection. Only in exceptional cases may additives be used with the methanol-water mixture, since additives will change the properties of the column. For chromatograms with additives it is mandatory to use a separate column of the same type. If methanol-water is not appropriate, other organic solvent-water mixtures call be used, e.g. ethanol-water or acetonitrile-water.

The pH of the eluent is critical for ionisable compounds. It should be within the operating pH range of the column, which is usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not advisable since the use of an alkaline, mobile phase may cause rapid deterioration in the performance of the column.

The reference compounds should be the purest available. Compounds to be used for test or calibration purposes are dissolved in the mobile phase if possible.

The temperature during the measurements should not vary by more than ± 2 K.

1.6.3.2.    Measurement

The dead time to can be determined by using either a homologous series (e.g. n-alkyl methyl ketones) or unretained organic compounds (e.g. thiourea or formamide). For calculating the dead time to by using a homologous series, a set of at least seven members of a homologous series is injected and the respective retention times are determined. The raw retention times tr (nc + 1) are plotted as a function of tr(nc) and the intercept a and slope b of the regression equation:

tr(nc + 1) = a + b tr(nc)

are determined (nc = number of carbon atoms). The dead time to is then given by:

to = a/(1 - b)

The next step is to construct a correlation plot of log k values versus log p for appropriate reference compounds. In practice, a set of between 5 and 10 standard reference compounds whose log p is around the expected range are injected simultaneously and the retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are calculated and plotted as a function of the log p determined by the shake-flask method. The calibration is performed at regular intervals, at least once daily, so that possible changes in column performance can be allowed for.

The test substance is injected in as small a quantity of mobile phase as possible. The retention time is determined (in duplicate), permitting the calculation of the capacity factor k. From the correlation graph of the reference compounds, the partition coefficient of the test substance can be interpolated. For very low and very high partition coefficients, extrapolation is necessary. In those cases particular care has to be taken of the confidence limits of the regression line.

2.   DATA

Shake-flask method

The reliability of the determined values of P can be tested by comparison of the means of the duplicate determinations with the overall mean.

3.   REPORTING

The test report shall, if possible, include the following information:

 precise specification of the substance (identity and impurities),

 when the methods are not applicable (e.g. surface active material), a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided,

 all information and remarks relevant for the interpretation of results, especially with regard to impurities and physical state of the substance.

For shake-flask method:

 the result of the preliminary estimation, if any,

 temperature of the determination,

 data on the analytical procedures used in determining concentrations,

 time and speed of centrifugation, if used,

 the measured concentrations in both phases for each determination (this means that a total of 12 concentrations will be reported),

 the weight of the test substance, the volume of each phase employed in each test vessel and the total calculated amount of test substance present in each phase after equilibration,

 the calculated values of the partition coefficient (P) and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the partition coefficient, this should be noted in the report,

 the standard deviation of individual P values about their mean should be reported,

 the mean P from all determinations should also be expressed as its logarithm (base 10),

 the calculated theoretical Pow when this value has been determined or when the measured value is > 104,

 pH of water used and of the aqueous phase during the experiment,

 if buffers are used, justification for the use of buffers in place of water, composition, concentration and pH of the buffers, pH of the aqueous phase before and after the experiment.

For HPLC method:

 the result of the preliminary estimation, if any,

 test and reference substances, and their purity,

 temperature range of the determinations,

 pH at which the determinations are made,

 details of the analytical and guard column, mobile phase and means of detection,

 retention data and literature log P values for reference compounds used in calibration,

 details of fitted regression line (log k versus log P),

 average retention data and interpolated log P value for the test compound,

 description of equipment and operating conditions,

 elution profiles,

 quantities of test and references substances introduced in the column,

 dead-time and how it was measured.

4.   REFERENCES

(1) OECD, Paris, 1981, Test Guideline 107, Decision of the Council C(81) 30 final.

(2) C. Hansch and A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.

(3) Log P and Parameter Database, A tool for the quantitative prediction of bioactivity (C. Hansch, chairman, A.J. Leo, dir.) — Available from Pomona College Medical Chemistry Project 1982, Pomona College, Claremont, California 91711.

(4) L. Renberg, G. Sundström and K. Sundh-Nygärd, Chemosphere, 1980, vol. 80, p. 683.

(5) H. Ellgehausen, C. D'Hondt and R. Fuerer, Pestic. Sci., 1981, vol. 12, p. 219.

(6) B. McDuffie, Chemosphere, 1981, vol. 10, p. 73.

(7) W.E. Hammers et al., J. Chromatogr., 1982, vol. 247, p. 1.

(8) J.E. Haky and A.M. Young, J. Liq. Chromat., 1984, vol. 7, p. 675.

(9) S. Fujisawa and E. Masuhara, J. Biomed. Mat. Res., 1981, vol. 15, p. 787.

(10) O. Jubermann, Verteilen und Extrahieren, in Methoden der Organischen Chemie (Houben Weyl), Allgemeine Laboratoriumpraxis (edited by E. Muller), Georg Thieme Verlag, Stuttgart, 1958, Band I/1, p. 223-339.

(11) R.F. Rekker and H.M. de Kort, Euro. J. Med. Chem., 1979, vol. 14, p. 479.

(12) A. Leo, C. Hansch and D. Elkins, Partition coefficients and their uses. Chem. Rev., 1971, vol. 71, p. 525.

(13) R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977.

(14) NF T 20-043 AFNOR (1985). Chemical products for industrial use — Determination of partition coefficient — Flask shaking method.

(15) C.V. Eadsforth and P. Moser, Chemosphere, 1983, vol. 12, p. 1459.

(16) A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, vol. 71, p. 525.

(17) C. Hansch, A. Leo, S.H. Unger, K.H. Kim, D. Nikaitani and E.J. Lien, J. Med. Chem., 1973, vol. 16, p. 1207.

(18) W.B. Neely, D.R. Branson and G.E. Blau, Environ. Sci. Technol., 1974, vol. 8, p. 1113.

(19) D.S. Brown and E.W. Flagg, J. Environ. Qual., 1981, vol. 10, p. 382.

(20) J.K. Seydel and K.J. Schaper, Chemische Struktur und biologische Aktivität von Wirkstoffen, Verlag Chemie, Weinheim, New York, 1979.

(21) R. Franke, Theoretical Drug Design Methods, Elsevier, Amsterdam, 1984.

(22) Y.C. Martin, Quantitative Drug Design, Marcel Dekker, New York, Base1, 1978.

(23) N.S. Nirrlees, S.J. Noulton, C.T. Murphy, P.J. Taylor; J. Med. Chem., 1976, vol. 19, p. 615.

Appendix 1

Calculation/estimation methods

INTRODUCTION

A general introduction to calculation methods, data and examples are provided in the Handbook of Chemical Property Estimation Methods (a).

Calculated values of Pow can be used:

 for deciding which of the experimental methods is appropriate (shake-flask range: log Pow: - 2 to 4, HPLC range: log Pow: 0 to 6),

 for selecting the appropriate test conditions (e.g. reference substances for HPLC procedures, volume ratio n-octanol/water for shake flask method),

 as a laboratory internal check on possible experimental errors,

 for providing a Pow-estimate in cases where the experimental methods cannot be applied for technical reasons.

ESTIMATION METHOD

Preliminary estimate of the partition coefficient

The value of the partition coefficient can be estimated by the use of the solubilities of the test substance in the pure solvents: For this:

image

CALCULATION METHODS

Principle of the calculation methods

All calculation methods are based on the formal fragmentation of the molecule into suitable substructures for which reliable log Pow-increments are known. The log Pow of the whole molecule is then calculated as the sum of its corresponding fragment values plus the sum of correction terms for intramolecular interactions.

Lists of fragment constants and correction terms ate available (b)(c)(d)(e);. Some are regularly updated (b).

Quality criteria

In general, the reliability of the calculation method decreases with increasing complexity of the compound under study. In the case of simple molecules with low molecular weight and one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured value can be expected. In the case of more complex molecules the margin of error can be greater. This will depend on the reliability and availability of fragment constants, as well as on the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of the correction terms (less of a problem with the computer software CLOGP-3) (b). In the case of ionising compounds the correct consideration of the charge or degree of ionisation is important.

Calculation procedures

The original hydrophobic substituent constant, π, introduced by Fujira et al. (f) is defined as:

πx = log Pow (PhX) - log Pow (PhH)

where Pow (PhX) is the partition coefficient of an aromatic derivative and Pow (PhH) that of the parent compound

(e.g. πCl = log Pow (C6H5Cl) - log Pow (C6H6) = 2,84 - 2,13 = 0,71 ).

According to its definition the π-method is applicable predominantly for aromatic substitution. π-values for a large number of substituents have been tabulated (b)(c)(d). They are used for the calculation of log Pow for aromatic molecules or substructures.

According to Rekker (g) the log Pow value is calculated as follows:

image

where fi represents the different molecular fragment constants and ai the frequency of their occurrence in the molecule under investigation. The correction terms can be expressed as an integral multiple of one single constant Cm (so-called magic constant). The fragment constants fi and Cm were determined from a list of 1 054 experimental Pow values (825 compounds) using multiple regression analysis (c)(h). The determination of the interaction terms is carried out according to set rules described in the literature (e)(h)(i).

According to Hansch and Leo (c), the log Pow value is calculated from:

image

where fi represents the different molecular fragment constants, Fj the correction terms and ai, bj the corresponding frequencies of occurrence. Derived from experimental Pow values, a list of atomic and group fragmental values and a list of correction terms Fj (so-called factors) were determined by trial and error. The correction terms have been ordered into several different classes (a)(c). It is relatively complicated and time consuming to take into account all the rules and correction terms. Software packages have been developed (b).

The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (b)(c) or from one's own measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch π-values or with Rekker or Leo fragment constants.

Remarks

(i) The calculation methods can only be applied to partly or fully ionised compounds when it is possible to take the necessary correction factors into account;

(ii) if intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. + 0,6 to + 1,0 log Pow units) have to be added (a). Indications for the presence of such bonds can be obtained from stereo models or spectroscopic data of the molecule;

(iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation;

(iv) the revisions of lists of fragment constants should be followed carefully.

Report

When using calculation/estimation methods, the test report shall, if possible, include the following information:

 description of the substance (mixture, impurities, etc.),

 indication of any possible intramolecular hydrogen bonding, dissociation, charge and any other unusual effects (e.g. tautomerism),

 description of the calculation method,

 identification or supply of database,

 peculiarities in the choice of fragments,

 comprehensive documentation of the calculation.

LITERATURE

(a) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.), Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, 1983.

(b) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

(c) C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.

(d) A. Leo, C. Hansch, D. Elkins, Chem. Rev., 1971, vol. 71, p. 525.

(e) R.F. Rekker, H.M. de Kort, Eur. J. Med. Chem. -Chill. Ther. 1979, vol. 14, p. 479.

(f) T. Fujita, J. Iwasa and C. Hansch, J. Amer. Chem. Soc., 1964, vol. 86, p. 5175.

(g) R.F. Rekker, The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Elsevier, New York, 1977, vol. 1.

(h) C.V. Eadsforth, P. Moser, Chemosphere, 1983, vol. 12, p. 1459.

(i) R.A. Scherrer, ACS, American Chemical Society, Washington D.C., 1984, Symposium Series 255, p. 225.

Appendix 2

Recommended Reference Substances for the HLPC Method



No

Reference Substance

log Pow

pKa

1

2-Butanone

0,3

 

2

4-Acetylpyridine

0,5

 

3

Aniline

0,9

 

4

Acetanilide

1,0

 

5

Benzylalcohol

1,1

 

6

p-Methoxyphenol

1,3

pKa = 10,26

7

Phenoxy acetic acid

1,4

pKa = 3,12

8

Phenol

1,5

pKa = 9,92

9

2,4-Dinitrophenol

1,5

pKa = 3,96

10

Benzonitrile

1,6

 

11

Phenylacetonitrile

1,6

 

12

4-Methylbenzyl alcohol

1,6

 

13

Acetophenone

1,7

 

14

2-Nitrophenol

1,8

pKa = 7,17

15

3-Nitrobenzoic acid

1,8

pKa = 3,47

16

4-Chloraniline

1,8

pKa = 4,15

17

Nitrobenzene

1,9

 

18

Cinnamic alcohol

1,9

 

19

Benzoic acid

1,9

pKa = 4,19

20

p-Cresol

1,9

pKa = 10,17

21

Cinnamic acid

2,1

pKa = 3,89 cis 4,44 trans

22

Anisole

2,1

 

23

Methylbenzoate

2,1

 

24

Benzene

2,1

 

25

3-Methylbenzoic acid

2,4

pKa = 4,27

26

4-Chlorophenol

2,4

pKa = 9,1

27

Trichloroethylene

2,4

 

28

Atrazine

2,6

 

29

Ethylbenzoate

2,6

 

30

2,6-Dichlorobenzonitrile

2,6

 

31

3-Chlorobenzoic acid

2,7

pKa = 3,82

32

Toluene

2,7

 

33

1-Naphthol

2,7

pKa = 9,34

34

2,3-Dichloroaniline

2,8

 

35

Chlorobenzene

2,8

 

36

Allyl-phenylether

2,9

 

37

Bromobenzene

3,0

 

38

Ethylbenzene

3,2

 

39

Benzophenone

3,2

 

40

4-Phenylphenol

3,2

pKa = 9,54

41

Thymol

3,3

 

42

1,4-Dichlorobenzene

3,4

 

43

Diphenylamine

3,4

pKa = 0,79

44

Naphthalene

3,6

 

45

Phenylbenzoate

3,6

 

46

Isopropylbenzene

3,7

 

47

2,4,6-Trichlorophenol

3,7

pKa = 6

48

Biphenyl

4,0

 

49

Benzylbenzoate

4,0

 

50

2,4-Dinitro-6 sec. butyophenol

4,1

 

51

1,2,4-Trichlorobenzene

4,2

 

52

Dodecanoic acid

4,2

 

53

Diphenylether

4,2

 

54

n-Butylbenzene

4,5

 

55

Phenanthrene

4,5

 

56

Fluoranthene

4,7

 

57

Dibenzyl

4,8

 

58

2,6-Diphenylpyridine

4,9

 

59

Triphenylamine

5,7

 

60

DDT

6,2

 

Other reference substances of low log Pow

1

Nicotinic acid

- 0,07

 

A.9.   FLASH-POINT

1.   METHOD

1.1.   INTRODUCTION

It is useful to have preliminary information on the flammability of the substance before performing this test. The test procedure is applicable to liquid substances whose vapours can be ignited by ignition sources. The test methods listed in this text are only reliable for flash-point ranges which are specified in the individual methods.

The possibility of chemical reactions between the substance and the sample holder should be considered when selecting the method to be used.

1.2.   DEFINITIONS AND UNITS

The flash-point is the lowest temperature, corrected to a pressure of 101,325 kPa, at which a liquid evolves vapours, under the conditions defined in the test method, in such an amount that a flammable vapour/air mixture is produced in the test vessel.

Units: oC

t = T - 273,15

(t in oC and T in K)

1.3.   REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4.   PRINCIPLE OF THE METHOD

The substance is placed in a test vessel and heated or cooled to the test temperature according to the procedure described in the individual test method. Ignition trials are carried out in order to ascertain whether or not the sample flashed at the test temperature.

1.5.   QUALITY CRITERIA

1.5.1.   Repeatability

The repeatability varies according to flash-point range and the test method used; maximum 2 oC.

1.5.2.   Sensitivity

The sensitivity depends on the test method used.

1.5.3.   Specificity

The specificity of some test methods is limited to certain flash-point ranges and subject to substance-related data (e.g. high viscosity).

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Preparations

A sample of the test substance is placed in a test apparatus according to 1.6.3.1 and/or 1.6.3.2.

For safety, it is recommended that a method utilising a small sample size, circa 2 cm3, be used for energetic or toxic substances.

1.6.2.   Test conditions

The apparatus should, as far as is consistent with safety, be placed in a draught-free position.

1.6.3.   Performance of the test

1.6.3.1.   Equilibrium method

See ISO 1516, ISO 3680, ISO 1523, ISO 3679.

1.6.3.2.   Non-equilibrium method

See BS 2000 part 170, NF M07-011, NF T66-009.

See EN 57, DIN 51755 part 1 (for temperatures from 5 to 65 oC), DIN 51755 part 2 (for temperatures below 5 oC), NF M07-036.

See ASTM D 56.

See ISO 2719, EN 11, DIN 51758, ASTM D 93, BS 2000-34, NF M07-019.

When the flash-point, determined by a non-equilibrium method in 1.6.3.2, is found to be 0 ± 2 oC, 21 ± 2 oC or 55 ± 2 oC, it should be confirmed by an equilibrium method using the same apparatus.

Only the methods which can give the temperature of the flash-point may be used for a notification.

To determine the flash-point of viscous liquids (paints, gums and similar) containing solvents, only apparatus and test methods suitable for determining the flash-point of viscous liquids may be used.

See ISO 3679, ISO 3680, ISO 1523, DIN 53213 part 1.

2.  DATA

3.   REPORTING

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 the method used should be stated as well as any possible deviations,

 the results and any additional remarks relevant for the interpretation of results.

4.   REFERENCES

None.

A.10.   FLAMMABILITY (SOLIDS)

1.   METHOD

1.1.   INTRODUCTION

It is useful to have preliminary information on potentially explosive properties of the substance before performing this test.

This test should only be applied to powdery, granular or paste-like substances.

In order not to include all substances which can be ignited but only those which burn rapidly or those whose burning behaviour is in any way especially dangerous, only substances whose burning velocity exceeds a certain limiting value are considered to be highly flammable.

It can be especially dangerous if incandescence propagates through a metal powder because of the difficulties in extinguishing a fire. Metal powders should be considered highly flammable if they support spread of incandescence throughout the mass within a specified time.

1.2.   DEFINITION AND UNITS

Burning time expressed in seconds.

1.3.   REFERENCE SUBSTANCES

Not specified.

1.4.   PRINCIPLE OF THE METHOD

The substance is formed into an unbroken strip or powder train about 250 mm long and a preliminary screening test performed to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation over 200 mm of the train occurs within a specified time then a full test programme to determine the burning rate is carried out.

1.5.   QUALITY CRITERIA

Not stated.

1.6.   DESCRIPTION OF METHOD

1.6.1.   Preliminary screening test

The substance is formed into an unbroken strip or powder train about 250 mm long by 20 mm wide by 10 mm high on a non-combustible, non-porous and low heat-conducting base plate. A hot flame from a gas burner (minimum diameter 5 mm) is applied to one end of the powder train until the powder ignites or for a maximum of two minutes (five minutes for powders of metals or metal-alloys). It should be noted whether combustion propagates along 200 mm of the train within the 4 minutes test period (or 40 minutes for metal powders). If the substance does not ignite and propagate combustion either by burning with flame or smouldering along 200 mm of the powder train within the four minutes (or 40 minutes) test period, then the substance should not be considered as highly flammable and no further testing is required. If the substance propagates burning of a 200 mm length of the powder train in less than four minutes, or less than 40 minutes for metal powders, the procedure described below (point 1.6.2. and following) should be carried out.

1.6.2.   Burning rate test

1.6.2.1.   Preparation

Powdery or granular substances are loosely filled into a mould 250 mm long with a triangular cross-section of inner height 10 mm and width 20 mm. On both sides of the mould in a longitudinal direction two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross section (figure). The mould is then dropped three times from a height of 2 cm onto a solid surface. If necessary the mould is then filled up again. The lateral limitations are then removed and the excess substance scraped off. A non-combustible, non-porous and low heat-conducting base plate is placed on top of the mould, the apparatus inverted and the mould removed.

Paste-like substances are spread on a non-combustible, non-porous and low heat-conducting base plate in the form of a rope 250 mm in length with a cross section of about 1 cm2.

1.6.2.2.   Test conditions

In the case a moisture-sensitive substance, the test should be carried out as quickly as possible after its removal from the container.

1.6.2.3.   Performance of the test

Arrange the pile across the draught in a fume cupboard.

The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus.

A hot flame from a gas burner (minimum diameter of 5 mm) is used to ignite the pile at one end. When the pile has burned a distance of 80 mm, the rate of burning over the next 100 mm is measured.

The test is performed six times, using a clean cool plate each time, unless a positive result is observed earlier.

2.   DATA

The burning time from the preliminary screening test (1.6.1) and the shortest burning time in up to six tests (1.6.2.3) are relevant for evaluation.

3.   REPORTING

3.1.   TEST REPORT

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 a description of the substance to be tested, its physical state including moisture content,

 results from the preliminary screening test and from the burning rate test if performed,

 all additional remarks relevant to the interpretation of results.

3.2.   INTERPRETATION OF THE RESULT

Powdery, granular or paste-1ike substances are to be considered as highly flammable when the time of burning in any tests carried out according to the test procedure described in 1.6.2 is less than 45 seconds. Powders of metals or metal-alloys are considered to be highly flammable when they can be ignited and the flame or the zone of reaction spreads over the whole sample in 10 minutes or less.

4.   REFERENCES

NF T 20-042 (September 85) Chemical products for industrial use. Determination of the flammability of solids.

Appendix

Figure

Mould and accessories for the preparation of the pile

(All dimensions in millimetres)

image

A.11.   FLAMMABILITY (GASES)

1.   METHOD

1.1.   INTRODUCTION

This method allows a determination of whether gases mixed with air at room temperature (circa 20 oC) and atmospheric pressure are flammable and, if so, over what range of concentrations. Mixtures of increasing concentrations of the test gas with air are exposed to an electrical spark and it is observed whether ignition occurs.

1.2.   DEFINITION AND UNITS

The range of flammability is the range of concentration between the lower and the upper explosion limits. The lower and the upper explosion limits are those limits of concentration of the flammable gas in admixture with air at which propagation of a flame does not occur.

1.3.   REFERENCE SUBSTANCES

Not specified.

1.4.   PRINCIPLE OF THE METHOD

The concentration of gas in air is increased step by step and the mixture is exposed at each stage to an electrical spark.

1.5.   QUALITY CRITERIA

Not stated.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Apparatus

The test vessel is an upright glass cylinder having a minimum inner diameter of 50 mm and a minimum height of 300 mm. The ignition electrodes are separated by a distance of 3 to 5 mm and are placed 60 mm above the bottom of the cylinder. The cylinder is fitted with a pressure-release opening. The apparatus has to be shielded to restrict any explosion damage.

A standing induction spark of 0,5 sec. duration, which is generated from a high voltage transformer with an output voltage of 10 to 15 kV (maximum of power input 300 W), is used as the ignition source. An example of a suitable apparatus is described in reference (2).

1.6.2.   Test conditions

The test must be performed at room temperature (circa 20 oC).

1.6.3.   Performance of the test

Using proportioning pumps, a known concentration of gas in air is introduced into the glass cylinder. A spark is passed through the mixture and it is observed whether or not a flame detaches itself from the ignition source and propagates independently. The gas concentration is varied in steps of 1 % vol. until ignition occurs as described above.

If the chemical structure of the gas indicates that it would be non-flammable and the composition of the stoichiometric mixture with air can be calculated, then only mixtures in the range from 10 % less than the stoichiometric composition to 10 % greater than this composition need be tested in 1 % steps.

2.   DATA

The occurrence of flame propagation is the only relevant information data for the determination of this property.

3.   REPORTING

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 a description, with dimensions, of the apparatus used,

 the temperature at which the test was performed,

 the tested concentrations and the results obtained,

 the result of the test: non-flammable gas or highly flammable gas,

 if it is concluded that the gas is non-flammable then the concentration range over which it was tested in 1 % steps should be stated,

 all information and remarks relevant to the interpretation of results have to be reported.

4.   REFERENCES

(1) NF T 20-041 (September 85) Chemical products for industrial use. Determination of the flammability of gases.

(2) W. Berthold, D. Conrad, T. Grewer, H. Grosse-Wortmann ‘Entwicklung einer Standard-Apparatur zur Messung von Explosionsgrenzen’. Chem.-Ing.- Tech. 1984, vo1. 56, 2, 126-127., T. Redeker und H. Schacke, p. 126-127.

A.12.   FLAMMABILITY (CONTACT WITH WATER)

1.   METHOD

1.1.   INTRODUCTION

This test method can be used to determine whether the reaction of a substance with water or damp air leads to the development of dangerous amounts of gas or gases which may be highly flammable.

The test method can be applied to both solid and liquid substances. This method is not applicable to substances which spontaneously ignite when in contact with air.

1.2.   DEFINITIONS AND UNITS

Highly flammable: substances which, in contact with water or damp air, evolve highly flammable gases in dangerous quantities at a minimum rate of 1 litre/kg per hour.

1.3.   PRINCIPLE OF THE METHOD

The substance is tested according to the step by step sequence described below; if ignition occurs at any step, no further testing is necessary. If it is known that the substance does not react violently with water then proceed to step 4 (1.3.4).

1.3.1.   Step 1

The test substance is placed in a trough containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites.

1.3.2.   Step 2

The test substance is placed on a filter paper floating on the surface of a dish containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites. The filter paper is merely to keep the substance in one place to increase the chances of ignition.

1.3.3.   Step 3

The test substance is made into a pile approximately 2 cm high and 3 cm diameter. A few drops of water are added to the pile and it is noted whether or not the evolved gas ignites.

1.3.4.   Step 4

The test substance is mixed with distilled water at 20 oC and the rate of evolution of gas is measured over a period of seven hours, at one-hour intervals. If the rate of evolution is erratic, or is increasing, after seven hours, the measuring time should be extended to a maximum time of five days. The test may be stopped if the rate at any time exceeds 1 litre/kg per hour.

1.4.   REFERENCE SUBSTANCES

Not specified.

1.5.   QUALITY CR1TERIA

Not stated.

1.6.   DESCRIPTION OF METHODS

1.6.1.   Step 1

1.6.1.1.   Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.1.2.   Performance of the test

A small quantity (approximately 2 mm diameter) of the test substance should be placed in a trough containing distilled water. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.2.   Step 2

1.6.2.1.   Apparatus

A filter-paper is floated flat on the surface of distilled water in any suitable vessel, e.g. a 100 mm diameter evaporating dish.

1.6.2.2.   Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.2.3.   Performance of the test

A small quantity of the test substance (approximately 2 mm diameter) is placed onto the centre of the filter-paper. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.3.   Step 3

1.6.3.1.   Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.3.2.   Performance of the test

The test substance is made into a pile approximately 2 cm high and 3 cm diameter with an indentation in the top. A few drops of water are added to the hollow and a note is made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.4.   Step 4

1.6.4.1.   Apparatus

The apparatus is set up as shown in the figure.

1.6.4.2.   Test conditions

Inspect the container of the test substance for any powder < 500 μm (particle size). If the powder constitutes more than 1 % w/w of the total, or if the sample is friable, then the whole of the substance should be ground to a powder before testing to allow for a reduction in particle size during storage and handling; otherwise the substance is to be tested as received. The test should be performed at room temperature (circa 20 oC) and atmospheric pressure.

1.6.4.3.   Performance of the test

10 to 20 ml of water are put into the dropping funnel of the apparatus and 10 g of substance are put in the conical flask. The volume of gas evolved can be measured by any suitable means. The tap of the dropping funnel is opened to let the water into the conical flask and a stop watch is started. The gas evolution is measured each hour during a seven hour period. If, during this period, the gas evolution is erratic, or if, at the end of this period, the rate of gas evolution is increasing, then measurements should be continued for up to five days. If, at any time of measurement, the rate of gas evolution exceeds 1 litre/kg per hour, the test can be discontinued. This test should be performed in triplicate.

If the chemical identity of the gas is unknown, the gas should be analysed. When the gas contains highly flammable components and it is unknown whether the whole mixture is highly flammable, a mixture of the same composition has to be prepared and tested according to the method A.11.

2.   DATA

The substance is considered hazardous if:

 spontaneous ignition takes place in any step of the test procedure,

 or

 there is evolution of flammable gas at a rate greater than 1 litre/kg of the substance per hour.

3.   REPORTING

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 details of any initial preparation of the test substance,

 the results of the tests (steps 1, 2, 3 and 4),

 the chemical identity of gas evolved,

 the rate of evolution of gas if step 4 (1.6.4) is performed,

 any additional remarks relevant to the interpretation of the results.

4.   REFERENCES

(1) Recommendations on the transport of dangerous goods, test and criteria, 1990, United Nations, New York.

(2) NF T 20-040 (September 85) Chemical products for industrial use. Determination of the flammability of gases formed by the hydrolysis of solid and liquid products.

Appendix

Figure

Apparatus

image

A.13.   PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

1.   METHOD

1.1.   INTRODUCTION

The test procedure is applicable to solid or liquid substances, which, in small amounts, will ignite spontaneously a short time after coming into contact with air at room temperature (circa 20 oC).

Substances which need to be exposed to air for hours or days at room temperature or at elevated temperatures before ignition occurs are not covered by this test method.

1.2.   DEFINITIONS AND UNITS

Substances are considered to have pyrophoric properties if they ignite or cause charring under the conditions described in 1.6.

The auto-flammability of liquids may also need to be tested using method A.15. Auto-ignition temperature (liquids and gases).

1.3.   REFERENCE SUBSTANCES

Not specified.

1.4.   PRINCIPLE OF THE METHOD

The substance, whether solid or liquid, is added to an inert carrier and brought into contact with air at ambient temperature for a period of five minutes. If liquid substances do not ignite then they are absorbed onto filter paper and exposed to air at ambient temperature (circa 20 oC) for five minutes. If a solid or liquid ignites, or a liquid ignites or chars a filter paper, then the substance is considered to be pyrophoric.

1.5.   QUALITY CRITERIA

Repeatability: because of the importance in relation to safety, a single positive result is sufficient for the substance to be considered pyrophoric.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Apparatus

A porcelain cup of circa 10 cm diameter is filled with diatomaceous earth to a height of about 5 mm at room temperature (circa 20 oC).

Note:

Diatomaceous earth or any other comparable inert substance which is generally obtainable shall be taken as representative of soil onto which the test substance might be spilled in the event of an accident.

Dry filter paper is required for testing liquids which do not ignite on contact with air when in contact with an inert carrier.

1.6.2.   Performance of the test

(a)   Powdery solids

1 to 2 cm3 of the substance to be tested is poured from circa 1 m height onto a non-combustible surface and it is observed whether the substance ignites during dropping or within five minutes of settling.

The test is performed six times unless ignition occurs;

(b)   liquids

Circa 5 cm3 of the liquid to be tested is poured into the prepared porcelain cup and it is observed whether the substance ignites within five minutes.

If no ignition occurs in the six tests, perform the following tests:

A 0,5 ml test sample is delivered from a syringe to an indented filter paper and it is observed whether ignition or charring of the filter paper occurs within five minutes of the liquid being added. The test is performed three times unless ignition or charring occurs.

2.   DATA

2.1.   TREATMENT OF RESULTS

Testing can be discontinued as soon as a positive result occurs in any of the tests.

2.2.   EVALUATION

If the substance ignites within five minutes when added to an inert carrier and exposed to air, or a liquid substance chars or ignites a filter paper within five minutes when added and exposed to air, it is considered to be pyrophoric.

3.   REPORTING

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 the results of the tests,

 any additional remark relevant to the interpretation of the results.

4.   REFERENCES

(1) NF T 20-039 (September 85) Chemical products for industrial use. Determination of the spontaneous flammability of solids and liquids.

(2) Recommendations on the Transport of Dangerous Goods, Test and criteria, 1990, United Nations, New York.

A.14.   EXPLOSIVE PROPERTIES

1.   METHOD

1.1.   INTRODUCTION

The method provides a scheme of testing to determine whether a solid or a pasty substance presents a danger of explosion when submitted to the effect of a flame (thermal sensitivity), or to shock or friction (sensitivity to mechanical stimuli), and whether a liquid substance presents a danger of explosion when submitted to the effect of a flame or shock.

The method comprises three parts:

(a) a test of thermal sensitivity (1);

(b) a test of mechanical sensitivity with respect to shock (1);

(c) a test of mechanical sensitivity with respect to friction (1).

The method yields data to assess the likelihood of initiating an explosion by means of certain common stimuli. The method is not intended to ascertain whether a substance is capable of exploding under any conditions.

The method is appropriate for determining whether a substance will present a danger of explosion (thermal and mechanical sensitivity) under the particular conditions specified in the directive. It is based on a number of types of apparatus which are widely used internationally (1) and which usually give meaningful results. It is recognised that the method is not definitive. Alternative apparatus to that specified may be used provided that it is internationally recognised and the results can be adequately correlated with those from the specified apparatus.

The tests need not be performed when available thermodynamic information (e.g. heat of formation, heat of decomposition) and/or absence of certain reactive groups (2) in the structural formula establishes beyond reasonable doubt that the substance is incapable of rapid decomposition with evolution of gases or release of heat (i.e. the material does not present any risk of explosion). A test of mechanical sensitivity with respect to friction is not required for liquids.

1.2.   DEFINITIONS AND UNITS

Explosive:

Substances which may explode under the effect of flame or which are sensitive to shock or friction in the specified apparatus (or are more mechanically sensitive than 1,3 -dinitrobenzene in alternative apparatus).

1.3.   REFERENCE SUBSTANCES

1,3-dinitrobenzene, technical crystalline product sieved to pass 0,5 mm, for the friction and shock methods.

Perhydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen, cyclonite — CAS 121-82-4), recrystallised from aqueous cyclohexanone, wet-sieved through a 250 μm and retained on a 150 μm sieve and dried at 103 ± 2 oC (for four hours) for the second series of friction and shock tests.

1.4.   PRINCIPLE OF THE METHOD

Preliminary tests are necessary to establish safe conditions for the performance of the three tests of sensitivity.

1.4.1.   Safety-in-handling tests (3)

For safety reasons, before performing the main tests, very small samples (circa 10 mg) of the substance are subjected to heating without confinement in a gas flame, to shock in any convenient form of apparatus and to friction by the use of a mallet against an anvil or any form of friction machine. The objective is to ascertain if the substance is so sensitive and explosive that the prescribed sensitivity tests, particularly that of thermal sensitivity, should be performed with special precautions so as to avoid injury to the operator.

1.4.2.   Thermal sensitivity

The method involves heating the substance in a steel tube, closed by orifice plates with differing diameters of hole, to determine whether the substance is liable to explode under conditions of intense heat and defined confinement.

1.4.3.   Mechanical sensitivity (shock)

The method involves subjecting the substance to the shock from a specified mass dropped from a specified height.

1.4.4.   Mechanical sensitivity (friction)

The method involves subjecting solid or pasty substances to friction between standard surfaces under specified conditions of load and relative motion.

1.5.   QUALITY CRITERIA

Not stated.

1.6.   DESCRIPTION OF METHOD

1.6.1.   Thermal sensitivity (effect of a flame)

1.6.1.1.   Apparatus

The apparatus consists of a non-reusable steel tube with its re-usable closing device (figure 1), installed in a heating and protective device. Each tube is deep-drawn from sheet steel (see Appendix) and has an internal diameter of 24 mm, a length of 75 mm and wall thickness of 0,5 mm. The tubes are flanged at the open end to enable them to be closed by the orifice plate assembly. This consists of a pressure-resistant orifice plate, with a central hole, secured firmly to a tube using a two-part screw joint (nut and threaded collar). The nut and threaded collar are made from chromium-manganese steel (see Appendix) which is spark-free up to 800 oC. The orifice plates are 6 mm thick, made from heat-resistant steel (see Appendix), and are available with a range of diameters of opening.

1.6.1.2.   Test conditions

Normally the substance is tested as received although in certain cases, e.g. if pressed, cast or otherwise condensed, it may be necessary to test the substance after crushing.

For solids, the mass of material to be used in each test is determined using a two-stage dry run procedure. A tared tube is filled with 9 cm3 of substance and the substance tamped with 80 N force applied to the total cross-section of the tube. For reasons of safety or in cases where the physical form of the sample can be changed by compression other filling procedures may be used; e.g. if the substance is very friction sensitive then tamping is not appropriate. If the material is compressible then more is added and tamped until the tube is filled to 55 mm from the top. The total mass used to fill the tube to the 55 mm level is determined and two further increments, each tamped with 80 N force, are added. Material is then either added with tamping, or taken out, as required, to leave the tube filled to a level 15 mm from the top. A second dry run is performed, starting with a tamped quantity of a third of the total mass found in the first dry run. Two more of these increments are added with 80 N tamping and the level of the substance in the tube adjusted to 15 mm from the top by addition or subtraction of material as required. The amount of solid determined in the second dry run is used for each trial; filling being performed in three equal amounts, each compressed to 9 cm3 by whatever force is necessary. (This may be facilitated by the use of spacing rings).

Liquids and gels are loaded into the tube to a height of 60 mm taking particular care with gels to prevent the formation of voids. The threaded collar is slipped onto the tube from below, the appropriate orifice plate is inserted and the nut tightened after applying some molybdenum disulphide based lubricant. It is essential to check that none of the substance is trapped between the flange and the plate, or in the threads.

Heating is provided by propane taken from an industrial cylinder, fitted with a pressure regulator (60 to 70 mbar), through a meter and evenly distributed (as indicated by visual observation of the flames from the burners) by a manifold to four burners. The burners are located around the test chamber as shown in figure 1. The four burners have a combined consumption of about 3,2 litres of propane per minute. Alternative fuel gases and burners may be used but the heating rate must be as specified in figure 3. For all apparatus, the heating rate must be checked periodically using tubes filled with dibutyl phthalate as indicated in figure 3.

1.6.1.3.   Performance of the tests

Each test is performed until either the tube is fragmented or the tube has been heated for five minutes. A test resulting in the fragmentation of the tube into three or more pieces, which in some cases may be connected to each other by narrow strips of metal as illustrated in figure 2, is evaluated as giving an explosion. A test resulting in fewer fragments or no fragmentation is regarded as not giving an explosion.

A series of three tests with a 6,0 mm diameter orifice plate is first performed and, if no explosions are obtained, a second series of three tests is performed with a 2,0 mm diameter orifice plate. If an explosion occurs during either test series then no further tests are required.

1.6.1.4.   Evaluation

The test result is considered positive if an explosion occurs in either of the above series of tests.

1.6.2.   Mechanical sensitivity (shock)

1.6.2.1.   Apparatus (figure 4)

The essential parts of a typical fall hammer apparatus are a cast steel block with base, anvil, column, guides, drop weights, release device and a sample holder. The steel anvil 100 mm (diameter) × 70 mm (height) is screwed to the top of a steel block 230 mm (length) × 250 mm (width) × 200 mm (height) with a cast base 450 mm (length) × 450 mm (width) × 60 mm (height). A column, made from seamless drawn steel tube, is secured in a holder screwed on to the back of the steel block. Four screws anchor the apparatus to a solid concrete block 60 × 60 × 60 cm such that the guide rails are absolutely vertical and the drop weight falls freely. 5 and 10 kg weights, made from solid steel, are available for use. The striking head of each weight is of hardened steel, HRC 60 to 63, and has a minimum diameter of 25 mm.

The sample under test is enclosed in a shock device consisting of two coaxial solid steel cylinders, one above the other, in a hollow cylindrical steel guide ring. The solid steel cylinders should be of 10 (- 0,003 , - 0,005 ) mm diameter and 10 mm height and have polished surfaces, rounded edges (radius of curvature 0,5 mm) and a hardness of HRC 58 to 65. The hollow cylinder must have an external diameter of 16 mm, a polished bore of 10 (+ 0,005 , + 0,010 ) mm and a height of 13 mm. The shock device is assembled on an intermediate anvil (26 mm diameter and 26 mm height) made of steel and centred by a ring with perforations to allow escape of fumes.

1.6.2.2.   Test conditions

The sample volume should be 40 mm3, or a volume to suit any alternative apparatus. Solid substances should be tested in the dry state and prepared as follows:

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction from 0,5 to 1 mm diameter is used for testing and should be representative of the original substance.

Substances normally supplied as pastes should be tested in the dry state where possible or, in any case, following removal of the maximum possible amount of diluent. Liquid substances are tested with a 1 mm gap between the upper and lower steel cylinders.

1.6.2.3.   Performance of the tests

A series of six tests are performed dropping the 10 kg mass from 0,40 m (40 J). If an explosion is obtained during the six tests at 40 J, a further series of six tests, dropping a 5 kg mass from 0,15 m (7,5 J), must be performed. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique etc.).

1.6.2.4.   Evaluation

The test result is considered positive if an explosion (bursting into flame and/or a report is equivalent to explosion) occurs at least once in any of the tests with the specified shock apparatus or the sample is more sensitive than 1,3-dinitrobenzene or RDX in an alternative shock test.

1.6.3.   Mechanical sensitivity (friction)

1.6.3.1.   Apparatus (figure 5)

The friction apparatus consists of a cast steel base plate on which is mounted the friction device. This consists of a fixed porcelain peg and moving porcelain plate. The porcelain plate is held in a carriage which runs in two guides. The carriage is connected to an electric motor via a connecting rod, an eccentric cam and suitable gearing such that the porcelain plate is moved, once only, back and forth beneath the porcelain peg for a distance of 10 mm. The porcelain peg may be loaded with, for example, 120 or 360 newtons.

The flat porcelain plates are made from white technical porcelain (roughness 9 to 32 μm) and have the dimensions 25 mm (length) × 25 mm (width) × 5 mm (height). The cylindrical porcelain peg is also made of white technical porcelain and is 15 mm long, has a diameter of 10 mm and roughened spherical end surfaces with a radius of curvature of 10 mm.

1.6.3.2.   Test conditions

The sample volume should be 10 mm3 or a volume to suit any alternative apparatus.

Solid substances are tested in the dry state and prepared as follows:

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction < 0,5 mm diameter is used for testing.

Substances normally supplied as pastes should be tested in the dry state where possible. If the substance cannot be prepared in the dry state, the paste (following removal of the maximum possible amount of diluent) is tested as a 0,5 mm thick, 2 mm wide, 10 mm long film, prepared with a former.

1.6.3.3.   Performance of the tests

The porcelain peg is brought onto the sample under test and the load applied. When carrying out the test, the sponge marks of the porcelain plate must lie transversely to the direction of the movement. Care must be taken that the peg rests on the sample, that sufficient test material lies under the peg and also that the plate moves correctly under the peg. For pasty substances, a 0,5 mm thick gauge with a 2 × 10 mm slot is used to apply the substance to the plate. The porcelain plate has to move 10 mm forwards and backwards under the porcelain peg in a time of 0,44 seconds. Each part of the surface of the plate and peg must only be used once; the two ends of each peg will serve for two trials and the two surfaces of a plate will each serve for three trials.

A series of six tests are performed with a 360 N loading. If a positive event is obtained during these six tests, a further series of six tests must be performed with a 120 N loading. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique, etc.).

1.6.3.4.   Evaluation

The test result is considered positive if an explosion (crepitation and/or a report or bursting into flame are equivalent to explosion) occurs at least once in any of the tests with the specified friction apparatus or satisfies the equivalent criteria in an alternative friction test.

2.   DATA

In principle, a substance is considered to present a danger of explosion in the sense of the directive if a positive result is obtained in the thermal, shock or friction sensitivity test.

3.   REPORTING

3.1.   TEST REPORT

The test report shall, if possible, include the following information:

 identity, composition, purity, moisture content, etc. of the substance tested,

 the physical form of the sample and whether or not it has been crushed, broken and/or sieved,

 observations during the thermal sensitivity tests (e.g. sample mass, number of fragments, etc.),

 observations during the mechanical sensitivity tests (e.g. formation of considerable amounts of smoke or complete decomposition without a report, flames, sparks, report, crepitation, etc.),

 results of each type of test,

 if alternative apparatus has been used, scientific justification as well as evidence of correlation between results obtained with specified apparatus and those obtained with equivalent apparatus must be given,

 any useful comments such as reference to tests with similar products which might be relevant to a proper interpretation of the results,

 all additional remarks relevant for the interpretation of the results.

3.2.   INTERPRETATION AND EVALUATION OF RESULTS

The test report should mention any results which are considered false, anomalous or unrepresentative. If any of the results should be discounted, an explanation and the results of any alternative or supplementary testing should be given. Unless an anomalous result can be explained, it must be accepted at face value and used to classify the substance accordingly.

4.   REFERENCES

(1) Recommendations on the Transport of Dangerous Goods: Tests and criteria, 1990, United Nations, New York.

(2) Bretherick, L., Handbook of Reactive Chemical Hazards, 4th edition, Butterworths, London, ISBN 0-750-60103-5, 1990.

(3) Koenen, H., Ide, K.H. and Swart, K.H., Explosivstoffe, 1961, vol. 3, 6-13 and 30-42.

(4) NF T 20-038 (September 85) Chemical products for industrial use — Determination of explosion risk.

Appendix

Example of material specification for thermal sensitivity test (see DIN 1623)

(1) Tube: Material specification No 1.0336.505 g

(2) Orifice plate: Material specification No 1.4873

(3) Threaded collar and nut: Material specification No 1.3817

Figure 1

Thermal sensitivity test apparatus

(all dimensions in millimetres)

image

Figure 2

Thermal sensitivity test

(example of fragmentation)

image

Figure 3

Heating rate calibration for thermal sensitivity test

image

Temperature/time curve obtained on heating dibutyl phtalate (27 cm3) in a closed (1,5 mm orifice plate) tube using a propane flow rate of 3,2 litre/minute. The temperature is measured with a 1 mm diameter stainless steel sheathed chromel/alumel thermocouple, placed centrally 43 mm below the rim of the tube. The heating rate between 135 oC and 285 oC should be between 185 and 215 K/minute.

Figure 4

Shock test apparatus

(all dimensions in millimetres)

image

Figure 4

Continued

image

Figure 5

Friction sensitivity apparatus

image

A.15.   AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

1.   METHOD

1.1.   INTRODUCTION

Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test. The test procedure is applicable to gases, liquids and vapours which, in the presence of air, can be ignited by a hot surface.

The auto-ignition temperature can be considerably reduced by the presence of catalytic impurities, by the surface material or by a higher volume of the test vessel.

1.2.   DEFINITIONS AND UNITS

The degree of auto-ignitability is expressed in terms of the auto-ignition temperature. The auto-ignition temperature is the lowest temperature at which the test substance will ignite when mixed with air under the conditions defined in the test method.

1.3.   REFERENCE SUBSTANCES

Reference substances are cited in the standards (see 1.6.3). They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4.   PRINCIPLE OF THE METHOD

The method determines the minimum temperature of the inner surface of an enclosure that will result in ignition of a gas, vapour or liquid injected into the enclosure.

1.5.   QUALITY CRITERIA

The repeatability varies according to the range of auto-ignition temperatures and the test method used.

The sensitivity and specificity depend on the test method used.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Apparatus

The apparatus is described in the method referred to in 1.6.3.

1.6.2.   Test conditions

A sample of the test substance is tested according to the method referred to in 1.6.3.

1.6.3.   Performance of the test

See IEC 79-4, DIN 51794, ASTM-E 659-78, BS 4056, NF T 20-037.

2.   DATA

Record the test-temperature, atmospheric pressure, quantity of sample used and time-1ag until ignition occurs.

3.   REPORTING

The test report shall, if possible, include the following information:

 the precise specification of the substance (identification and impurities),

 the quantity of sample used, atmospheric pressure,

 the apparatus used,

 the results of measurements (test temperatures, results concerning ignition, corresponding time-lags),

 all additional remarks relevant to the interpretation of results.

4.   REFERENCES

None.

A.16.   RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

1.   METHOD

1.1.   INTRODUCTION

Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test.

The purpose of this test is to provide preliminary information on the auto-flammability of solid substances at elevated temperatures.

If the heat developed either by a reaction of the substance with oxygen or by exothermic decomposition is not lost rapidly enough to the surroundings, self-heating leading to self-ignition occurs. Self-ignition therefore occurs when the rate of heat-production exceeds the rate of heat loss.

The test procedure is useful as a preliminary screening test for solid substances. In view of the complex nature of the ignition and combustion of solids, the self-ignition temperature determined according to this test method should be used for comparison purposes only.

1.2.   DEFINITIONS AND UNITS

The self-ignition temperature as obtained by this method is the minimum ambient temperature expressed in oC at which a certain volume of a substance will ignite under defined conditions.

1.3.   REFERENCE SUBSTANCE

None.

1.4.   PRINCIPLE OF THE METHOD

A certain volume of the substance under test is placed in an oven at room temperature; the temperature/time curve relating to conditions in the centre of the sample is recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min. For the purpose of this test, the temperature of the oven at which the sample temperature reaches 400 oC by self-heating is called the self-ignition temperature.

1.5.   QUALITY CRITERIA

None.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Apparatus

1.6.1.1.   Oven

A temperature-programmed laboratory oven (volume about 2 litres) fitted with natural air circulation and explosion relief. In order to avoid a potential explosion risk, any decomposition gases must not be allowed to come into contact with the electric heating elements.

1.6.1.2.   Wire mesh cube

A piece of stainless steel wire mesh with 0,045 mm openings should be cut according to the pattern in figure 1. The mesh should be folded and secured with wire into an open-topped cube.

1.6.1.3.   Thermocouples

Suitable thermocouples.

1.6.1.4.   Recorder

Any two-channel recorder calibrated from 0 to 600 oC or corresponding voltage.

1.6.2.   Test conditions

Substances are tested as received.

1.6.3.   Performance of the test

The cube is filled with the substance to be tested and is tapped gently, adding more of the substance until the cube is completely full. The cube is then suspended in the centre of the oven at room temperature. One thermocouple is placed at the centre of the cube and the other between the cube and the oven wall to record the oven temperature.

The temperatures of the oven and sample are continuously recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min.

When the substance ignites the sample thermocouple will show a very sharp temperature rise above the oven temperature.

2.   DATA

The temperature of the oven at which the sample temperature reaches 400 oC by self-heating is relevant for evaluation (see figure 2).

3.   REPORTING

The test report shall, if possible, include the following information:

 a description of the substance to be tested,

 the results of measurement including the temperature/time curve,

 all additional remarks relevant for the interpretation of the results.

4.   REFERENCES

NF T 20-036 (September 85) Chemical products for industrial use. Determination of the relative temperature of the spontaneous flammability of solids.

Figure 1

Pattern of 20 mm test cube

image

Figure 2

Typical temperature/time curve

image

A.17.   OXIDISING PROPERTIES (SOLIDS)

1.   METHOD

1.1.   INTRODUCTION

It is useful to have preliminary information on any potentially explosive properties of the substance before performing this test.

This test is not applicable to liquids, gases, explosive or highly flammable substances, or organic peroxides.

This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.

In order to ascertain if the test should be performed with special precautions, a preliminary test should be performed.

1.2.   DEFINITION AND UNITS

Burning time: reaction time, in seconds, taken for the reaction zone to travel along a pile, following the procedure described in 1.6.

Burning rate: expressed in millimetres per second.

Maximum burning rate: the highest value of the burning rates obtained with mixtures containing 10 to 90 % by weight of oxidiser.

1.3.   REFERENCE SUBSTANCE

Barium nitrate (analytical grade) is used as reference substance for the test and the preliminary test.

The reference mixture is that mixture of barium nitrate with powdered cellulose, prepared according to 1.6, which has the maximum burning rate (usually a mixture with 60 % barium nitrate by weight).

1.4.   PRINCIPLE OF THE METHOD

A preliminary test is carried out in the interests of safety. No further testing is required when the preliminary test clearly indicates that the test substance has oxidising properties. When this is not the case, the substance should then be subject to the full test.

In the full test, the substance to be tested and a defined combustible substance will be mixed in various ratios. Each mixture is then formed into a pile and the pile is ignited at one end. The maximum burning rate determined is compared with the maximum burning rate of the reference mixture.

1.5.   QUALITY CRITERIA

If required, any method of grinding and mixing is valid provided that the difference in the maximum rate of burning in the six separate tests differs from the arithmetic mean value by no more than 10 %.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Preparation

1.6.1.1.   Test substance

Reduce the test sample to a particle size < 0,125 mm using the following procedure: sieve the test substance, grind the remaining fraction, repeat the procedure until the whole test portion has passed the sieve.

Any grinding and sieving method satisfying the quality criteria may be used.

Before preparing the mixture the substance is dried at 105 oC, until constant weight is obtained. If the decomposition temperature of the substance to be tested is below 105 oC, the substance has to be dried at a suitable lower temperature.

1.6.1.2.   Combustible substance

Powdered cellulose is used as a combustible substance. The cellulose should be a type used for thin-layer chromatography or column chromatography. A type with fibre-lengths of more than 85 % between 0,020 and 0,075 mm has proved to be suitable. The cellulose powder is passed through a sieve with a mesh-size of 0,125 mm. The same batch of cellulose is to be used throughout the test.

Before preparing the mixture, the powdered cellulose is dried at 105 oC until constant weight is obtained.

If wood-meal is used in the preliminary test, then prepare a soft-wood wood-meal by collecting the portion which passes through a sieve mesh of 1,6 mm, mix thoroughly, then dry at 105 oC for four hours in a layer not more than 25 mm thick. Cool and store in an air-tight container filled as full as practicable until required, preferably within 24 hours of drying.

1.6.1.3.   Ignition source

A hot flame from a gas burner (minimum diameter 5 mm) should be used as the ignition source. If another ignition source is used (e.g. when testing in an inert atmosphere), the description and the justification should be reported.

1.6.2.   Performance of the test

Note:

Mixtures of oxidisers with cellulose or wood-meal must be treated as potentially explosive and handled with due care.

1.6.2.1.   Preliminary test

The dried substance is thoroughly mixed with the dried cellulose or wood-meal in the proportions 2 of test substance to 1 of cellulose or wood-meal by weight and the mixture is formed into a small cone-shaped pile of dimensions 3,5 cm (diameter of base) × 2,5 cm (height) by filling, without tamping, a cone-shaped former (e.g. a laboratory glass funnel with the stem plugged).

The pile is placed on a cool, non-combustible, non-porous and low heat-conducting base plate. The test should be carried out in a fume cupboard as in 1.6.2.2.

The ignition source is put in contact with the cone. The vigour and duration of the resultant reaction are observed and recorded.

The substance is to be considered as oxidising if the reaction is vigorous.

In any case where the result is open to doubt, it is then necessary to complete the full train test described below.

1.6.2.2.   Train test

Prepare oxidiser cellulose-mixtures containing 10 to 90 % weight of oxidiser in 10 % increments. For borderline cases, intermediate oxidiser cellulose mixtures should be used to obtain the maximum burning rate more precisely.

The pile is formed by means of a mould. The mould is made of metal, has a length of 250 mm and a triangular cross-section with an inner height of 10 mm and an inner width of 20 mm. On both sides of the mould, in the longitudinal direction, two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross-section (figure). This arrangement is loosely filled with a slight excess of mixture. After dropping the mould once from a height of 2 cm onto a solid surface, the remaining excess substance is scraped off with an obliquely positioned sheet. The lateral limitations are removed and the remaining powder is smoothed, using a roller. A non-combustible, non-porous and low heat-conducting base plate is then placed on the top of the mould, the apparatus inverted and the mould removed.

Arrange the pile across the draught in a fume cupboard.

The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus.

Due to hygroscopicity of cellulose and of some substances to be tested, the test should be carried out as quickly as possible.

Ignite one end of the pile by touching with the flame.

Measure the time of reaction over a distance of 200 mm after the reaction zone has propagated an initial distance of 30 mm.

The test is performed with the reference substance and at least once with each one of the range of mixtures of the test substance with cellulose.

If the maximum burning rate is found to be significantly greater than that from the reference mixture, the test can be stopped; otherwise the test should be repeated five times for each of the three mixtures giving the fastest burning rate.

If the result is suspected of being a false positive, then the test should be repeated using an inert substance with a similar particle size, such as kieselguhr, in place of cellulose. Alternatively, the test substance cellulose mixture, having the fastest burning rate, should be retested in an inert atmosphere (< 2 % v/v oxygen content).

2.   DATA

For safety reasons the maximum burning rate — not the mean value — shall be considered to be the characteristic oxidising property of the substance under test.

The highest value of burning rate within a run of six tests of a given mixture is relevant for evaluation.

Plot a graph of the highest value of burning rate for each mixture versus the oxidiser concentration. From the graph take the maximum burning rate.

The six measured values of burning rate within a run obtained from the mixture with the maximum burning rate must not differ from the arithmetic mean value by more than 10 %; otherwise the methods of grinding and mixing must be improved.

Compare the maximum burning rate obtained with the maximum burning rate of the reference mixture (see 1.3).

If tests are conducted in an inert atmosphere, the maximum reaction rate is compared with that from the reference mixture in an inert atmosphere.

3.   REPORT

3.1.   TEST REPORT

The test report shall, if possible, include the following information:

 the identity, composition, purity, moisture content etc. of the substance tested,

 any treatment of the test sample (e.g. grinding, drying),

 the ignition source used in the tests,

 the results of measurements,

 the mode of reaction (e.g. flash burning at the surface, burning through the whole mass, any information concerning the combustion products, etc.),

 all additional remarks relevant for the interpretation of results, including a description of the vigour (flaming, sparking, fuming, slow smouldering, etc.) and approximate duration produced in the preliminary safety/screening test for both test and reference substance,

 the results from tests with an inert substance, if any,

 the results from tests in an inert atmosphere, if any.

3.2.   INTERPRETATION OF THE RESULT

A substance is to be considered as an oxidising substance when:

(a) in the preliminary test, there is a vigorous reaction;

(b) in the full test, the maximum burning rate of the mixtures tested is higher than or equal to the maximum burning rate of the reference mixture of cellulose and barium nitrate.

In order to avoid a false positive, the results obtained when testing the substance mixed with an inert material and/or when testing under an inert atmosphere should also be considered when interpreting the results.

4.   REFERENCES

NF T 20-035 (September 85) Chemical products for industrial use. Determination of the oxidising properties of solids.

Appendix

Figure

Mould and accessories for the preparations of the pile

(All dimensions in millimetres)

image

A.18.   NUMBER-AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

1.   METHOD

This Gel Permeation Chromatographic method is a replicate of the OECD TG 118 (1996). The fundamental principles and further technical information are given in reference (1).

1.1.   INTRODUCTION

Since the properties of polymers are so varied, it is impossible to describe one single method setting out precisely the conditions for separation and evaluation which cover all eventualities and specificities occurring in the separation of polymers. In particular, complex polymer systems are often not amenable to gel permeation chromatography (GPC). When GPC is not practicable, the molecular weight may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the method used.

The method described is based on DIN Standard 55672 (1). Detailed information about how to carry out the experiments and how to evaluate the data can be found in this DIN Standard. In case modifications of the experimental conditions are necessary, these changes must be justified. Other standards may be used, if fully referenced. The method described uses polystyrene samples of known polydispersity for calibration and it may have to be modified to be suitable for certain polymers, e.g. water soluble and long-chain branched polymers.

1.2.   DEFINITIONS AND UNITS

The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations:



image

image

where,

Hi is the level of the detector signal from the baseline for the retention volume Vi,

Mi is the molecular weight of the polymer fraction at the retention volume Vi, and

n is the number of data points.

The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.3.   REFERENCE SUBSTANCES

Since GPC is a relative method, calibration must be undertaken. Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights Mn and Mw and a known molecular weight distribution are normally used for this. The calibration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical manner.

A determined relationship between the molecular weight and elution volume is only valid under the specific conditions of the particular experiment. The conditions include, above all, the temperature, the solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns.

The molecular weights of the sample determined in this way are relative values and are described as ‘polystyrene equivalent molecular weights’. This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a greater or a lesser degree. If other standards are used, e.g. polyethylene glycol, polyethylene oxide, polymethyl methacrylate, polyacrylic acid, the reason should be stated.

1.4.   PRINCIPLE OF THE TEST METHOD

Both the molecular weight distribution of the sample and the average molecular weights (Mn, Mw) can be determined using GPC. GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2).

Separation is effected as the sample passes through a column which is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are excluded. The path of the large molecules is thereby shorter and these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can penetrate all of the pores. These are eluted last.

In an ideal situation, the separation is governed entirely by the size of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and dead volumes can worsen the situation (2).

Detection is effected by, e.g. refractive index or UV-absorption, and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight.

1.5.   QUALITY CRITERIA

The repeatability (Relative Standard Deviation: RSD) of the elution volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are:



Mp < 2 000

Mw/Mn < 1,20

2 000 ≤ Mp ≤ 106

Mw/Mn < 1,05

Mp > 106

Mw/Mn < 1,20

(Mp is the molecular weight of the standard at the peak maximum)

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Preparation of the standard polystyrene solutions

The polystyrene standards are dissolved by careful mixing in the chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions.

The concentrations of the standards chosen are dependent on various factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. Typical injection volumes for analytical separations using GPC with a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. Higher volumes are possible, but they should not exceed 250 μl. The optimal ratio between the injection volume and the concentration must be determined prior to the actual calibration of the column.

1.6.2.   Preparation of the sample solution

In principle, the same requirements apply to the preparation of the sample solutions. The sample is dissolved in a suitable solvent, e.g. tetrahydrofuran (THF), by shaking carefully. Under no circumstances should it be dissolved using an ultrasonic bath. When necessary, the sample solution is purified via a membrane filter with a pore size of between 0,2 and 2 μm.

The presence of undissolved particles must be recorded in the final report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by weight of the undissolved particles. The solutions should be used within 24 hours.

1.6.3.   Apparatus

 solvent reservoir,

 degasser (where appropriate),

 pump,

 pulse dampener (where appropriate),

 injection system,

 chromatography columns,

 detector,

 flowmeter (where appropriate),

 data recorder-processor,

 waste vessel.

It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

1.6.4.   Injection and solvent delivery system

A defined volume of the sample solution is loaded onto the column either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.

1.6.5.   Column

Depending on the sample, the polymer is characterised using either a simple column or several columns connected in sequence. A number of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1)(2)(3).

1.6.6.   Theoretical plates

The column or the combination of columns used for separation must be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known length. The number of theoretical plates is given by the following equation:



image

or

image

where,

N

=

the number of theoretical plates

Ve

=

the elution volume at the peak maximum

W

=

the baseline peak width

W1/2

=

the peak width at half height

1.6.7.   Separation efficiency

In addition to the number of theoretical plates, which is a quantity determining the bandwidth, a part is also played by the separation efficiency, this being determined by the steepness of the calibration curve. The separation efficiency of a column is obtained from the following relationship:

image

where,

Ve, Mx

=

the elution volume for polystyrene with the molecular weight Mx

Ve,(10.Mx)

=

the elution volume for polystyrene with a ten times greater molecular weight

The resolution of the system is commonly defined as follows:

image

where,

Ve1, Ve2

=

the elution volumes of the two polystyrene standards at the peak maximum

W1, W2

=

the peak widths at the base-line

M1, M2

=

the molecular weights at the peak maximum (should differ by a factor of 10)

The R-value for the column system should be greater than 1.7 (4).

1.6.8.   Solvents

All solvents must be of high purity (for THF purity of 99,5 % is used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is transported to the column via the pump.

1.6.9.   Temperature control

The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.10.   Detector

The purpose of the detector is to record quantitatively the concentration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be larger than 10 μl except for light scattering and viscosity detectors. Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, viscosity detectors, etc.

2.   DATA AND REPORTING

2.1.   DATA

The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data.

For each sample, two independent experiments must be carried out. They have to be analysed individually.

Mn, Mw, Mw/Mn and Mp must be provided for every measurement. It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used.

After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The number average and the weight-average molecular weights are generally determined by means of electronic data processing, based on the formulas of section 1.2. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3).

The distribution curve must be provided in the form of a table or as figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.

2.2.   TEST REPORT

The test report must include the following information:

2.2.1.   Test substance:

 available information about test substance (identity, additives, impurities),

 description of the treatment of the sample, observations, problems.

2.2.2.   Instrumentation:

 reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

 pump, pulse dampener, injection system,

 separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

 number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

 information on symmetry of the peaks,

 column temperature, kind of temperature control,

 detector (measurement principle, type, cuvette volume),

 flowmeter if used (manufacturer, measurement principle),

 system to record and process data (hardware and software).

2.2.3.   Calibration of the system:

 detailed description of the method used to construct the calibration curve,

 information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

 information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

 all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

 

 name of the sample,

 manufacturer of the sample,

 characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

 injection volume and injection concentration,

 Mp value used for calibration,

 elution volume or corrected retention time measured at the peak maxima,

 Mp calculated at the peak maximum,

 percentage error of the calculated Mp and the calibration value.

2.2.4.   Evaluation:

 evaluation on a time basis: methods used to ensure the required reproducibility (method of correction, internal standard, etc.),

 information about whether the evaluation was effected on the basis of the elution volume or the retention time,

 information about the limits of the evaluation if a peak is not completely analysed,

 description of smoothing methods, if used,

 preparation and pre-treatment procedures of the sample,

 the presence of undissolved particles, if any,

 injection volume (μl) and injection concentration (mg/ml),

 observations indicating effects which lead to deviations from the ideal GPC profile,

 detailed description of all modifications in the testing procedures,

 details of the error ranges,

 any other information and observations relevant for the interpretation of the results.

3.   REFERENCES

(1) DIN 55672(1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1.

(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds., (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons.

(3) ASTM D 3536-91, (1991). Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC) American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) ASTM D 5296-92, (1992) Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

Appendix

Examples of other methods for determination of number average molecular weight (Mn) for polymers

Gel permeation chromatography (GPC) is the preferred method for determination of Mn, especially when a set of standards are available, whose structure are comparable with the polymer structure. However, where there are practical difficulties in using GPC or there is already an expectation that the substance will fail a regulatory Mn criterion (and which needs confirming), alternative methods are available, such as:

1.   Use of colligative properties

1.1. Ebullioscopy/Cryoscopy

involves measurement of boiling point elevation (ebullioscopy) or freezing point depression (cryoscopy) of a solvent, when the polymer is added. The method relies on the fact that the effect of the dissolved polymer on the boiling/freezing point of the liquid is dependent on the molecular weight of the polymer (1) (2).

Applicability, Mn < 20 000 .

1.2. Lowering of vapour pressure

involves the measurement of the vapour pressure of a chosen reference liquid before and after the addition of known quantities of polymer (1) (2).

Applicability, Mn < 20 000 (theoretically; in practice however of limited value).

1.3 Membrane osmometry

relies on the principle of osmosis, i.e. the natural tendency of solvent molecules to pass through a semi-permeable membrane from a dilute to a concentrated solution to achieve equilibrium. In the test, the dilute solution is at zero concentration, whereas the concentrated solution contains the polymer. The effect of drawing solvent through the membrane causes a pressure differential that is dependent on the concentration and the molecular weight of the polymer (1) (3) (4).

Applicability, Mn between 20 000 - 200 000 .

1.4 Vapour phase osmometry

involves comparison of the rate of evaporation of a pure solvent aerosol to at least three aerosols containing the polymer at different concentrations (1)(2)(4).

Applicability, Mn < 20 000 .

2.   End-group analysis

To use this method, knowledge of both the overall structure of the polymer and the nature of the chain terminating end groups is needed (which must be distinguishable from the main skeleton by, e.g. NMR or titration/derivatisation). The determination of the molecular concentration of the end groups present on the polymer can lead to a value for the molecular weight (7) (8) (9).

Applicability, Mn up to 50 000 (with decreasing reliability).

3.   References

(1) Billmeyer, F.W. Jr., (1984) Textbook of Polymer Science, 3rd Edn., John Wiley, New York.

(2) Glover, C.A., (1975) Absolute Colligative Property Methods. Chapter 4. In: Polymer Molecular Weights, Part I P.E. Slade, Jr. ed., Marcel Dekker, New York.

(3) ASTM D 3750-79, (1979) Standard Practice for Determination of Number-Average Molecular Weight of Polymers by Membrane Osmometry. American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) Coll, H. (1989) Membrane Osmometry. In: Determination of Molecular Weight, A.R. Cooper ed., J. Wiley and Sons, pp. 25-52.

(5) ASTM 3592-77, (1977) Standard Recommended Practice for Determination of Molecular Weight by Vapour Pressure, American Society for Testing and Materials, Philadelphia, Pennsylvania.

(6) Morris, C.E.M., (1989) Vapour Pressure Osmometry. In: Determinationn of Molecular Weight, A.R. Cooper ed., John Wiley and Sons.

(7) Schröder, E., Müller, G., and Arndt, K-F., (1989) Polymer Characterisation, Carl Hanser Verlag, Munich.

(8) Garmon, R.G., (1975) End-Group Determinations, Chapter 3 In: Polymer Molecular Weights, Part I, P.E. Slade, Jr. ed., Marcel Dekker, New York.

(9) Amiya, S., et al. (1990) Pure and Applied Chemistry, 62, 2139-2146.

A.19.   LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

1.   METHOD

This Gel Permeation Chromatographic method is a replicate of the OECD TG 119 (1996). The fundamental principles and further technical information are given in the references.

1.1.   INTRODUCTION

Since the properties of polymers are so varied, it is impossible to describe one single method setting out precisely the conditions for separation and evaluation which cover all eventualities and specificities occurring in the separation of polymers. In particular, complex polymer systems are often not amenable to gel permeation chromatography (GPC). When GPC is not practicable, the molecular weight may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the method used.

The method described is based on DIN Standard 55672 (1). Detailed information about how to carry out the experiments and how to evaluate the data can be found in this DIN Standard. In case modifications of the experimental conditions are necessary, these changes must be justified. Other standards may be used, if fully referenced. The method described uses polystyrene samples of known polydispersity for calibration and it may have to be modified to be suitable for certain polymers, e.g. water soluble and long-chain branched polymers.

1.2.   DEFINITIONS AND UNITS

Low molecular weight is arbitrarily defined as a molecular weight below 1 000 dalton.

The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations:



image

image

where,

Hi

=

the level of the detector signal from the baseline for the retention volume Vi,

Mi

=

the molecular weight of the polymer fraction at the retention volume Vi, and n is the number of data points

The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.3.   REFERENCE SUBSTANCES

Since GPC is a relative method, calibration must be undertaken. Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights Mn and Mw and a known molecular weight distribution are normally used for this. The calibration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical manner.

A determined relationship between the molecular weight and elution volume is only valid under the specific conditions of the particular experiment. The conditions include, above all, the temperature, the solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns.

The molecular weights of the sample determined in this way are relative values and are described as ‘polystyrene equivalent molecular weights’. This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a greater or a lesser degree. If other standards are used, e.g. polyethylene glycol, polyethylene oxide, polymethyl methacrylate, polyacrylic acid, the reason should be stated.

1.4.   PRINCIPLE OF THE TEST METHOD

Both the molecular weight distribution of the sample and the average molecular weights (Mn, Mw) can be determined using GPC. GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2).

Separation is effected as the sample passes through a column which is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are excluded. The path of the large molecules is thereby shorter and these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can penetrate all of the pores. These are eluted last.

In an ideal situation, the separation is governed entirely by the size of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and dead volumes can worsen the situation (2).

Detection is effected by e.g. refractive index or UV-absorption and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure, e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight.

The low molecular weight content is derived from this curve. The calculation can only be accurate if the low molecular weight species respond equivalently on a per mass basis to the polymer as a whole.

1.5.   QUALITY CRITERIA

The repeatability (Relative Standard Deviation: RSD) of the elution volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are:



Mp < 2 000

Mw/Mn < 1,20

2 000 < Mp < 106

Mw/Mn < 1,05

Mp > 106

Mw/Mn < 1,20

(Mp is the molecular weight of the standard at the peak maximum)

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Preparation of the standard polystyrene solutions

The polystyrene standards are dissolved by careful mixing in the chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions.

The concentrations of the standards chosen are dependent on various factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. Typical injection volumes for analytical separations using GPC with a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. Higher volumes are possible, but they should not exceed 250 μl. The optimal ratio between the injection volume and the concentration must be determined prior to the actual calibration of the column.

1.6.2.   Preparation of the sample solution

In principle, the same requirements apply to the preparation of the sample solutions. The sample is dissolved in a suitable solvent, e.g. tetrahydrofuran (THF), by shaking carefully. Under no circumstances should it be dissolved using an ultrasonic bath. When necessary, the sample solution is purified via a membrane filter with a pore size of between 0,2 and 2 μm.

The presence of undissolved particles must be recorded in the final report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by weight of the undissolved particles. The solutions should be used within 24 hours.

1.6.3.   Correction for content of impurities and additives

Correction of the content of species of M < 1 000 for the contribution from non-polymer specific components present (e.g. impurities and/or additives) is usually necessary, unless the measured content is already < 1 %. This is achieved by direct analysis of the polymer solution or the GPC eluate.

In cases where the eluate, after passage through the column, is too dilute for a further analysis it must be concentrated. It may be necessary to evaporate the eluate to dryness and dissolve it again. Concentration of the eluate must be effected under conditions which ensure that no changes occur in the eluate. The treatment of the eluate after the GPC step is dependent on the analytical method used for the quantitative determination.

1.6.4.   Apparatus

GPC apparatus comprises the following components:

 solvent reservoir,

 degasser (where appropriate),

 pump,

 pulse dampener (where appropriate),

 injection system,

 chromatography columns,

 detector,

 flowmeter (where appropriate),

 data recorder-processor,

 waste vessel.

It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

1.6.5.   Injection and solvent delivery system

A defined volume of the sample solution is loaded onto the column either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.

1.6.6.   Column

Depending on the sample, the polymer is characterised using either a simple column or several columns connected in sequence. A number of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1) (2) (3).

1.6.7.   Theoretical plates

The column or the combination of columns used for separation must be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known length. The number of theoretical plates is given by the following equation:



image

or

image

where,

N

=

the number of theoretical plates

Ve

=

the elution volume at the peak maximum

W

=

the baseline peak width

W1/2

=

the peak width at half height

1.6.8.   Separation efficiency

In addition to the number of theoretical plates, which is a quantity determining the bandwidth, a part is also played by the separation efficiency, this being determined by the steepness of the calibration curve. The separation efficiency of a column is obtained from the following relationship:

image

where,

Ve, Mx

=

the elution volume for polystyrene with the molecular weight Mx

Ve,(10.Mx)

=

the elution volume for polystyrene with a ten times greater molecular weight

The resolution of the system is commonly defined as follows:

image

where,

Ve1, Ve2

=

the elution volumes of the two polystyrene standards at the peak maximum

W1, W2

=

the peak widths at the base-1ine

M1, M2

=

the molecular weights at the peak maximum (should differ by a factor of 10).

The R-value for the column system should be greater than 1,7 (4).

1.6.9.   Solvents

All solvents must be of high purity (for THF purity of 99,5 % is used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is transported to the column via the pump.

1.6.10.   Temperature control

The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.11.   Detector

The purpose of the detector is to record quantitatively the concentration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be larger than 10 μl except for light scattering and viscosity detectors. Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, viscosity detectors, etc.

2.   DATA AND REPORTING

2.1.   DATA

The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data.

For each sample, two independent experiments must be carried out. They have to be analysed individually. In all cases it is essential to determine also data from blanks, treated under the same conditions as the sample.

It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used.

After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The portion of the curve corresponding to molecular weights below 1 000 is determined and corrected as necessary for impurities and additives. The elution curves are generally evaluated by means of electronic data processing. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3).

If any insoluble polymer is retained on the column, its molecular weight is likely to be higher than that of the soluble fraction, and if not considered would result in an overestimation of the low molecular weight content. Guidance for correcting the low molecular weight content for insoluble polymer is provided in the Appendix.

The distribution curve must be provided in the form of a table or as figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.

2.2.   TEST REPORT

The test report must include the following information:

2.2.1.   Test substance:

 available information about test substance (identity, additives, impurities),

 description of the treatment of the sample, observations, problems.

2.2.2.   Instrumentation:

 reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

 pump, pulse dampener, injection system,

 separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

 number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

 information on symmetry of the peaks,

 column temperature, kind of temperature control,

 detector (measurement principle, type, cuvette volume),

 flowmeter if used (manufacturer, measurement principle),

 system to record and process data (hardware and software).

2.2.3.   Calibration of the system:

 detailed description of the method used to construct the calibration curve,

 information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

 information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

 all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

 

 name of the sample,

 manufacturer of the sample,

 characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

 injection volume and injection concentration,

 Mp value used for calibration,

 elution volume or corrected retention time measured at the peak maxima,

 Mp calculated at the peak maximum,

 percentage error of the calculated Mp and the calibration value.

2.2.4.   Information on the low molecular weight polymer content:

 description of the methods used in the analysis and the way in which the experiments were conducted,

 information about the percentage of the low molecular weight species content (w/w) related to the total sample,

 information about impurities, additives and other non-polymer species in percentage by weight related to the total sample.

2.2.5.   Evaluation:

 evaluation on a time basis: all methods to ensure the required reproducibility (method of correction, internal standard etc.),

 information about whether the evaluation was effected on the basis of the elution volume or the retention time,

 information about the limits of the evaluation if a peak is not completely analysed,

 description of smoothing methods, if used,

 preparation and pre-treatment procedures of the sample,

 the presence of undissolved particles, if any,

 injection volume (μl) and injection concentration (mg/ml),

 observations indicating effects which lead to deviations from the ideal GPC profile,

 detailed description of all modifications in the testing procedures,

 details of the error ranges,

 any other information and observations relevant for the interpretation of the results.

3.   REFERENCES

(1) DIN 55672 (1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1.

(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds. (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons.

(3) ASTM D 3536-91, (1991) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC). American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) ASTM D 5296-92, (1992) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

Appendix

Guidance for correcting low molecular content for the presence of insoluble polymer

When insoluble polymer is present in a sample, it results in mass loss during the GPC analysis. The insoluble polymer is irreversibly retained on the column or sample filter while the soluble portion of the sample passes through the column. In the case where the refractive index increment (dn/dc) of the polymer can be estimated or measured, one can estimate the sample mass lost on the column. In that case, one makes a correction using an external calibration with standard materials of known concentration and dn/dc to calibrate the response of the refractometer. In the example hereafter a poly(methyl methacrylate) (pMMA) standard is used.

In the external calibration for analysis of acrylic polymers, a pMMA standard of known concentration in tetrahydrofuran, is analysed by GPC and the resulting data are used to find the refractometer constant according to the equation:

K = R/(C × V × dn/dc)

where:

K

=

the refractometer constant (in microvolt second/ml),

R

=

the response of the pMMA standard (in microvolt/second),

C

=

the concentration of the pMMA standard (in mg/ml),

V

=

the injection volume (in ml), and

dn/dc

=

the refractive index increment for pMMA in tetrahydrofuran (in ml/mg).

The following data are typical for a pMMA standard:

R

=

2 937 891

C

=

1,07 mg/ml

V

=

0,1 ml

dn/dc

=

9 × 10-5 ml/mg

The resulting K value, 3,05 × 1011 is then used to calculate the theoretical detector response if 100 % of the polymer injected had eluted through the detector.

A.20.   SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

1.   METHOD

The method described is a replicate of the revised version of OECD TG 120 (1997). Further technical information is given in reference (1).

1.1.   INTRODUCTION

For certain polymers, such as emulsion polymers, initial preparatory work may be necessary before the method set out hereafter can be used. The method is not applicable to liquid polymers and to polymers that react with water under the test conditions.

When the method is not practical or not possible, the solution/extraction behaviour may be investigated by means of other methods. In such cases, full details and justification should be given for the method used.

1.2.   REFERENCE SUBSTANCES

None.

1.3.   PRINCIPLE OF THE TEST METHOD

The solution/extraction behaviour of polymers in an aqueous medium is determined using the flask method (see A.6 Water Solubility, Flask method) with the modifications described below.

1.4.   QUALITY CRITERIA

None.

1.5.   DESCRIPTION OF THE TEST METHOD

1.5.1.   Equipment

The following equipment is required for the method:

 crushing device, e.g. grinder for the production of particles of known size,

 apparatus for shaking with possibility of temperature control,

 membrane filter system,

 appropriate analytical equipment,

 standardised sieves.

1.5.2.   Sample preparation

A representative sample has first to be reduced to a particle size between 0,125 and 0,25 mm using appropriate sieves. Cooling may be required for the stability of the sample or for the grinding process. Materials of a rubbery nature can be crushed at liquid nitrogen temperature (1).

If the required particle size fraction is not attainable, action should be taken to reduce the particle size as much as possible, and the result reported. In the report, it is necessary to indicate the way in which the crushed sample was stored prior to the test

1.5.3.   Procedure

Three samples of 10 g of the test substance are weighed into each of three vessels fitted with glass stoppers and 1 000 ml of water is added to each vessel. If handling an amount of 10 g polymer proves impracticable, the next highest amount which can be handled should be used and the volume of water adjusted accordingly.

The vessels are tightly stoppered and then agitated at 20 oC. A shaking or stirring device capable of operating at constant temperature should be used. After a period of 24 hours, the content of each vessel is centrifuged or filtered and the concentration of polymer in the clear aqueous phase is determined by a suitable analytical method. If suitable analytical methods for the aqueous phase are not available, the total solubility/extractivity can be estimated from the dry weight of the filter residue or centrifuged precipitate.

It is usually necessary to differentiate quantitatively between the impurities and additives on the one hand and the low molecular weight species on the other hand. In the case of gravimetric determination, it is also important to perform a blank run using no test substance in order to account for residues arising from the experimental procedure.

The solution/extraction behaviour of polymers in water at 37 oC at pH 2 and pH 9 may be determined in the same way as described for the conduct of the experiment at 20 oC. The pH values can be achieved by the addition of either suitable buffers or appropriate acids or bases such as hydrochloric acid, acetic acid, analytical grade sodium or potassium hydroxide or NH3.

Depending on the method of analysis used, one or two tests should be performed. When sufficiently specific methods are available for direct analysis of the aqueous phase for the polymer component, one test as described above should suffice. However, when such methods are not available and determination of the solution/extraction behaviour of the polymer is limited to indirect analysis by determining only the total organic carbon content (TOC) of the aqueous extract, an additional test should be conducted. This additional test should also be done in triplicate, using ten times smaller polymer samples and the same amounts of water as those used in the first test.

1.5.4.   Analysis

1.5.4.1.   Test conducted with one sample size

Methods may be available for direct analysis of polymer components in the aqueous phase. Alternatively, indirect analysis of dissolved/extracted polymer components, by determining the total content of soluble parts and correcting for non polymer-specific components, could also be considered.

Analysis of the aqueous phase for the total polymeric species is possible:

either by a sufficiently sensitive method, e.g.:

 TOC using persulphate or dichromate digestion to yield CO2 followed by estimation by IR or chemical analysis,

 Atomic Absorption Spectrometry (AAS) or its Inductively Coupled Plasma (ICP) emission equivalent for silicon or metal containing polymers,

 UV absorption or spectrofluorimetry for aryl polymers,

 LC-MS for low molecular weight samples,

or by vacuum evaporation to dryness of the aqueous extract and spectroscopic (IR, UV, etc.) or AAS/ICP analysis of the residue.

If analysis of the aqueous phase as such is not practicable, the aqueous extract should be extracted with a water-immiscible organic solvent e.g. a chlorinated hydrocarbon. The solvent is then evaporated and the residue analysed as above for the notified polymer content. Any components in this residue which are identified as being impurities or additives are to be subtracted for the purpose of determining the degree of solution/extraction of the polymer itself.

When relatively large quantities of such materials are present, it may be necessary to subject the residue to e.g. HPLC or GC analysis to differentiate the impurities from the monomer and monomer-derived species present so that the true content of the latter can be determined.

In some cases, simple evaporation of the organic solvent to dryness and weighing the dry residue may be sufficient.

1.5.4.2.   Test conducted with two different sample sizes

All aqueous extracts are analysed for TOC.

A gravimetric determination is performed on the undissolved/not extracted part of the sample. If, after centrifugation or filtering of the content of each vessel, polymer residues remain attached to the wall of the vessel, the vessel should be rinsed with the filtrate until the vessel is cleared from all visible residues. Following which, the filtrate is again centrifuged or filtered. The residues remaining on the filter or in the centrifuge tube are dried at 40 oC under vacuum and weighed. Drying is continued until a constant weight is reached.

2.   DATA

2.1.   TEST CONDUCTED WITH ONE SAMPLE SIZE

The individual results for each of the three flasks and the average values should be given and expressed in units of mass per volume of the solution (typically mg/l) or mass per mass of polymer sample (typically mg/g). Additionally, the weight loss of the sample (calculated as the weight of the solute divided by the weight of the initial sample) should also be given. The relative standard deviations (RSD) should be calculated. Individual figures should be given for the total substance (polymer + essential additives, etc.) and for the polymer only (i.e. after subtracting the contribution from such additives).

2.2.   TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES

The individual TOC values of the aqueous extracts of the two triplicate experiments and the average value for each experiment should be given expressed as units of mass per volume of solution (typically mgC/l), as well as in units of mass per weight of the initial sample (typically mgC/g).

If there is no difference between the results at the high and the low sample/water ratios, this may indicate that all extractable components were indeed extracted. In such a case, direct analysis would normally not be necessary.

The individual weights of the residues should be given and expressed in percentage of the initial weights of the samples. Averages should be calculated per experiment. The differences between 100 and the percentages found represent the percentages of soluble and extractable material in the original sample.

3.   REPORTING

3.1.   TEST REPORT

The test report must include the following information:

3.1.1.   Test substance:

 available information about test substance (identity, additives, impurities, content of low molecular weight species).

3.1.2.   Experimental conditions:

 description of the procedures used and experimental conditions,

 description of the analytical and detection methods.

3.1.3.   Results:

 results of solubility/extractivity in mg/l; individual and mean values for the extraction tests in the various solutions, broken down in polymer content and impurities, additives, etc.,

 results of solubility/extractivity in mg/g of polymer,

 TOC values of aqueous extracts, weight of the solute and calculated percentages, if measured,

 the pH of each sample,

 information about the blank values,

 where necessary, references to the chemical instability of the test substance, during both the testing process and the analytical process,

 all information which is important for the interpretation of the results.

4.   REFERENCES

(1) DIN 53733 (1976) Zerkleinerung von Kunststofferzeugnissen für Prüfzwecke.

A.21.   OXIDISING PROPERTIES (LIQUIDS)

1.   METHOD

1.1.   INTRODUCTION

This test method is designed to measure the potential for a liquid substance to increase the burning rate or burning intensity of a combustible substance, or to form a mixture with a combustible substance which spontaneously ignites, when the two are thoroughly mixed. It is based on the UN test for oxidising liquids (1) and is equivalent to it. However, as this method A.21 is primarily designed to satisfy the requirements of Regulation (EC) No 1907/2006, comparison with only one reference substance is required. Testing and comparison to additional reference substances may be necessary when the results of the test are expected to be used for other purposes. ( 3 )

This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.

It is useful to have preliminary information on any potential explosive properties of the substance before performing this test.

This test is not applicable to solids, gases, explosive or highly flammable substances, or organic peroxides.

This test may not need to be performed when results for the test substance in the UN test for oxidising liquids (1) are already available.

1.2.   DEFINITIONS AND UNITS

Mean pressure rise time is the mean of the measured times for a mixture under test to produce a pressure rise from 690 kPa to 2 070 kPa above atmospheric.

1.3.   REFERENCE SUBSTANCE

65 % (w/w) aqueous nitric acid (analytical grade) is required as a reference substance. ( 4 )

Optionally, if the experimenter foresees that the results of this test may eventually be used for other purposes ( 5 ), testing of additional reference substances may also be appropriate. ( 6 )

1.4.   PRINCIPLE OF THE TEST METHOD

The liquid to be tested is mixed in a 1 to 1 ratio, by mass, with fibrous cellulose and introduced into a pressure vessel. If during mixing or filling spontaneous ignition occurs, no further testing is necessary.

If spontaneous ignition does not occur the full test is carried out. The mixture is heated in a pressure vessel and the mean time taken for the pressure to rise from 690 kPa to 2 070 kPa above atmospheric is determined. This is compared with the mean pressure rise time for the 1:1 mixture of the reference substance(s) and cellulose.

1.5.   QUALITY CRITERIA

In a series of five trials on a single substance no results should differ by more than 30 % from the arithmetic mean. Results that differ by more than 30 % from the mean should be discarded, the mixing and filling procedure improved and the testing repeated.

1.6.   DESCRIPTION OF THE METHOD

1.6.1.   Preparation

1.6.1.1.   Combustible substance

Dried, fibrous cellulose with a fibre length between 50 and 250 μm and a mean diameter of 25 μm ( 7 ), is used as the combustible material. It is dried to constant weight in a layer not more than 25 mm thick at 105 oC for four hours and kept in a desiccator, with desiccant, until cool and required for use. The water content of the dried cellulose should be less than 0,5 % by dry mass ( 8 ). If necessary, the drying time should be prolonged to achieve this. ( 9 ) The same batch of cellulose is to be used throughout the test.

1.6.1.2.   Apparatus

1.6.1.2.1.    Pressure vessel

A pressure vessel is required. The vessel consists of a cylindrical steel pressure vessel 89 mm in length and 60 mm in external diameter (see figure 1). Two flats are machined on opposite sides (reducing the cross-section of the vessel to 50 mm) to facilitate holding whilst fitting up the firing plug and vent plug. The vessel, which has a bore of 20 mm diameter is internally rebated at either end to a depth of 19 mm and threaded to accept 1'' British Standard Pipe (BSP) or metric equivalent. A pressure take-off, in the form of a side arm, is screwed into the curved face of the pressure vessel 35 mm from one end and at 90o to the machined flats. The socket for this is bored to a depth of 12 mm and threaded to accept the 1/2" BSP (or metric equivalent) thread on the end of the side-arm. If necessary, an inert seal is fitted to ensure a gas-tight seal. The side-arm extends 55 mm beyond the pressure vessel body and has a bore of 6 mm. The end of the side-arm is rebated and threaded to accept a diaphragm type pressure transducer. Any pressure-measuring device may be used provided that it is not affected by the hot gases or the decomposition products and is capable of responding to rates of pressure rise of 690-2 070 kPa in not more than 5 ms.

The end of the pressure vessel farthest from the side-arm is closed with a firing plug which is fitted with two electrodes, one insulated from, and the other earthed to, the plug body. The other end of the pressure vessel is closed by a bursting disk (bursting pressure approximately 2 200 kPa) held in place with a retaining plug which has a 20 mm bore. If necessary, an inert seal is used with the firing plug to ensure a gas-tight fit. A support stand (figure 2) holds the assembly in the correct attitude during use. This usually comprises a mild steel base plate measuring 235 mm × 184 mm × 6 mm and a 185 mm length of square hollow section (S.H.S.) 70 mm × 70 mm × 4 mm.

A section is cut from each of two opposite sides at one end of the length of S.H.S. so that a structure having two flat sided legs surmounted by 86 mm length of intact box section results. The ends of these flat sides are cut to an angle of 60o to the horizontal and welded to the base plate. A slot measuring 22 mm wide × 46 mm deep is machined in one side of the upper end of the base section such that when the pressure vessel assembly is lowered, firing plug end first, into the box section support, the side-arm is accommodated in the slot. A piece of steel 30 mm wide and 6 mm thick is welded to the lower internal face of the box section to act as a spacer. Two 7 mm thumb screws, tapped into the opposite face, serve to hold the pressure vessel firmly in place. Two 12 mm wide strips of 6 mm thick steel, welded to the side pieces abutting the base of the box section, support the pressure vessel from beneath.

1.6.1.2.2.    Ignition system

The ignition system consists of a 25 cm long Ni/Cr wire with a diameter 0,6 mm and a resistance of 3,85 ohm/m. The wire is wound, using a 5 mm diameter rod, in the shape of a coil and is attached to the firing plug electrodes. The coil should have one of the configurations shown in figure 3. The distance between the bottom of the vessel and the underside of the ignition coil should be 20 mm. If the electrodes are not adjustable, the ends of the ignition wire between the coil and the bottom of the vessel should be insulated by a ceramic sheath. The wire is heated by a constant current power supply able to deliver at least 10 A.

1.6.2.   Performance of the test ( 10 )

The apparatus, assembled complete with pressure transducer and heating system but without the bursting disk in position, is supported firing plug end down. 2,5 g of the liquid to be tested is mixed with 2,5 g of dried cellulose in a glass beaker using a glass stirring rod ( 11 ). For safety, the mixing should be performed with a safety shield between the operator and mixture. If the mixture ignites during mixing or filling, no further testing is necessary. The mixture is added, in small portions with tapping, to the pressure vessel making sure that the mixture is packed around the ignition coil and is in good contact with it. It is important that the coil is not distorted during the packing process as this may lead to erroneous results ( 12 ). The bursting disk is placed in position and the retaining plug is screwed in tightly. The charged vessel is transferred to the firing support stand, bursting disk uppermost, which should be located in a suitable, armoured fume cupboard or firing cell. The power supply is connected to the external terminals of the firing plug and 10 A applied. The time between the start of mixing and switching on the power should not exceed 10 minutes.

The signal produced by the pressure transducer is recorded on a suitable system which allows both evaluation and the generation of a permanent record of the time pressure profile obtained (e.g. a transient recorder coupled to a chart recorder). The mixture is heated until the bursting disk ruptures or until at least 60 s have elapsed. If the bursting disk does not rupture, the mixture should be allowed to cool before carefully dismantling the apparatus, taking precautions to allow for any pressurisation which may occur. Five trials are performed with the test substance and the reference substance(s). The time taken for the pressure to rise from 690 kPa to 2 070 kPa above atmospheric is noted. The mean pressure rise time is calculated.

In some cases, substances may generate a pressure rise (too high or too low), caused by chemical reactions not characterising the oxidising properties of the substance. In these cases, it may be necessary to repeat the test with an inert substance, e.g. diatomite (kieselguhr), in place of the cellulose in order to clarify the nature of the reaction.

2.   DATA

Pressure rise times for both the test substance and the reference substance(s). Pressure rise times for the tests with an inert substance, if performed.

2.1.   TREATMENT OF RESULTS

The mean pressure rise times for both the test substance and the reference substances(s) are calculated.

The mean pressure rise time for the tests with an inert substance (if performed) is calculated.

Some examples of results are shown in Table 1.



Table 1

Examples of results ()

Substance ()

Mean pressure rise time for a 1:1 mixture with celulose

(ms)

Ammonium dichromate, saturated aqueous solution

20 800

Calcium nitrate, saturated aqueous solution

6 700

Ferric nitrate, saturated aqueous solution

4 133

Lithium perchlorate, saturated aqueous solution

1 686

Magnesium perchlorate, saturated aqueous solution

777

Nickel nitrate, saturated aqueous solution

6 250

Nitric acid, 65 %

4 767  ()

Perchloric acid, 50 %

121 ()

Perchloric acid, 55 %

59

Potassium nitrate, 30 % aqueous solution

26 690

Silver nitrate, saturated aqueous solution

 ()

Sodium chlorate, 40 % aqueous solution

2 555  ()

Sodium nitrate, 45 % aqueous solution

4 133

Inert substance

 

Water: cellulose

 ()

(1)   See reference (1) for classification under the UN transport scheme.

(2)   Saturated solutions should be prepared at 20 oC.

(3)   Mean value from interlaboratory comparative trials.

(4)   Maximum pressure of 2 070 kPa not reached.

3.   REPORT

3.1.   TEST REPORT

The test report should include the following information:

 the identity, composition, purity, etc. of the substance tested,

 the concentration of the test substance,

 the drying procedure of the cellulose used,

 the water content of the cellulose used,

 the results of the measurements,

 the results from tests with an inert substance, if any,

 the calculated mean pressure rise times,

 any deviations from this method and the reasons for them,

 all additional information or remarks relevant to the interpretation of the results.

3.2.   INTERPRETATION OF THE RESULTS ( 13 )

The test results are assessed on the basis of:

(a) whether the mixture of test substance and cellulose spontaneously ignites; and

(b) the comparison of the mean time taken for the pressure to rise from 690 kPa to 2 070 kPa with that of the reference substance(s).

A liquid substance is to be considered as an oxidiser when:

(a) a 1:1 mixture, by mass, of the substance and cellulose spontaneously ignites; or

(b) a 1:1 mixture, by mass, of the substance and cellulose exhibits a mean pressure rise time less than or equal to the mean pressure rise time of a 1:1 mixture, by mass, of 65 % (w/w) aqueous nitric acid and cellulose.

In order to avoid a false positive result, if necessary, the results obtained when testing the substance with an inert material should also be considered when interpreting the results.

4.   REFERENCES

(1) Recommendations on the Transport of Dangerous Goods, Manual of Tests and Criteria. 3rd revised edition. UN Publication No: ST/SG/AC.10/11/Rev. 3, 1999, page 342. Test O.2: Test for oxidising liquids.

Figure 1

Pressure vessel

image

Figure 2

Support stand

image

Figure 3

Ignition system

image

Note: either of these configurations may be used.

▼M1

A.22.   LENGTH WEIGHTED GEOMETRIC MEAN DIAMETER OF FIBRES

1.   METHOD

1.1.   INTRODUCTION

This method describes a procedure to measure the Length Weighted Geometric Mean Diameter (LWGMD) of bulk Man Made Mineral Fibres (MMMF). As the LWGMD of the population will have a 95 % probability of being between the 95 % confidence levels (LWGMD ± two standard errors) of the sample, the value reported (the test value) will be the lower 95 % confidence limit of the sample (i.e. LWGMD — 2 standard errors). The method is based on an update (June 1994) of a draft HSE industry procedure agreed at a meeting between ECFIA and HSE at Chester on 26/9/93 and developed for and from a second inter-laboratory trial (1, 2). This measurement method can be used to characterise the fibre diameter of bulk substances or products containing MMMFs including refractory ceramic fibres (RCF), man-made vitreous fibres (MMVF), crystalline and polycrystalline fibres.

Length weighting is a means of compensating for the effect on the diameter distribution caused by the breakage of long fibres when sampling or handling the material. Geometric statistics (geometric mean) are used to measure the size distribution of MMMF diameters because these diameters usually have size distributions that approximate to log normal.

Measuring length as well as diameter is both tedious and time consuming but, if only those fibres that touch an infinitely thin line on a SEM field of view are measured, then the probability of selecting a given fibre is proportional to its length. As this takes care of the length in the length weighting calculations, the only measurement required is the diameter and the LWGMD-2SE can be calculated as described.

1.2.   DEFINITIONS

Particle: An object with a length to width ratio of less than 3:1.

Fibre: An object with a length to with ratio (aspect ratio) of at least 3:1.

1.3.   SCOPE AND LIMITATIONS

The method is designed to look at diameter distributions which have median diameters from 0,5 μm to 6 μm. Larger diameters can be measured by using lower SEM magnifications but the method will be increasingly limited for finer fibre distributions and a TEM (transmission electron microscope) measurement is recommended if the median diameter is below 0,5 μm.

1.4.   PRINCIPLE OF THE TEST METHOD

A number of representative core samples are taken from the fibre blanket or from loose bulk fibre. The bulk fibres are reduced in length using a crushing procedure and a representative sub-sample dispersed in water. Aliquots are extracted and filtered through a 0,2 μm pore size, polycarbonate filter and prepared for examination using scanning electron microscope (SEM) techniques. The fibre diameters are measured at a screen magnification of × 10 000 or greater ( 14 ) using a line intercept method to give an unbiased estimate of the median diameter. The lower 95 % confidence interval (based on a one sided test) is calculated to give an estimate of the lowest value of the geometric mean fibre diameter of the material.

1.5.   DESCRIPTION OF THE TEST METHOD

1.5.1.   Safety/precautions

Personal exposure to airborne fibres should be minimised and a fume cupboard or glove box should be used for handling the dry fibres. Periodic personal exposure monitoring should be carried out to determine the effectiveness of the control methods. When handling MMMF’s disposable gloves should be worn to reduce skin irritation and to prevent cross-contamination.

1.5.2.   Apparatus/equipment

 Press and dyes (capable of producing 10 MPa).

 0,2 μm pore size polycarbonate capillary pore filters (25 mm diameter).

 5 μm pore size cellulose ester membrane filter for use as a backing filter.

 Glass filtration apparatus (or disposable filtration systems) to take 25 mm diameter filters (e.g. Millipore glass microanalysis kit, type No XX10 025 00).

 Freshly distilled water that has been filtered through a 0,2 μm pore size filter to remove micro-organisms.

 Sputter coater with a gold or gold/palladium target.

 Scanning electron microscope capable of resolving down to 10 nm and operating at × 10 000 magnification.

 Miscellaneous: spatulas, type 24 scalpel blade, tweezers, SEM tubes, carbon glue or carbon adhesive tape, silver dag.

 Ultrasonic probe or bench top ultrasonic bath.

 Core sampler or cork borer, for taking core samples from MMMF blanket.

1.5.3.   Test Procedure

1.5.3.1.   Sampling

For blankets and bats a 25 mm core sampler or cork borer is used to take samples of the cross-section. These should be equally spaced across the width of a small length of the blanket or taken from random areas if long lengths of the blanket are available. The same equipment can be used to extract random samples from loose fibre. Six samples should be taken when possible, to reflect spatial variations in the bulk material.

The six core samples should be crushed in a 50 mm diameter dye at 10 MPa. The material is mixed with spatula and re-pressed at 10 MPa. The material is then removed from the dye and stored in a sealed glass bottle.

1.5.3.2.   Sample Preparation

If necessary, organic binder can be removed by placing the fibre inside a furnace at 450 °C for about one hour.

Cone and quarter to subdivide the sample (this should be done inside a dust cupboard).

Using a spatula, add a small amount (< 0,5 g) of sample to 100 ml of freshly distilled water that has been filtered through a 0,2 μm membrane filter (alternative sources of ultra pure water may be used if they are shown to be satisfactory). Disperse thoroughly by the use of an ultrasonic probe operated at 100 W power and tuned so that cavitation occurs. (If a probe is not available use the following method: repeatedly shake and invert for 30 seconds; ultrasonic in a bench top ultrasonic bath for five minutes; then repeatedly shake and invert for a further 30 seconds.)

Immediately after dispersion of the fibre, remove a number of aliquots (e.g. three aliquots of 3, 6 and 10 ml) using a wide-mouthed pipette (2-5 ml capacity).

Vacuum filter each aliquot through a 0,2 μm polycarbonate filter supported by a 5 μm pore MEC backing filter, using a 25 mm glass filter funnel with a cylindrical reservoir. Approximately 5 ml of filtered distilled water should be placed into the funnel and the aliquot slowly pipetted into the water holding the pipette tip below the meniscus. The pipette and the reservoir must be flushed thoroughly after pipetting, as thin fibres have a tendency to be located more on the surface.

Carefully remove the filter and separate it from the backing filter before placing it in a container to dry.

Cut a quarter or half filter section of the filtered deposit with a type 24 scalpel blade using a rocking action. Carefully attach the cut section to a SEM stub using a sticky carbon tab or carbon glue. Silver dag should be applied in at least three positions to improve the electrical contact at the edges of the filter and the stub. When the glue/silver dag is dry, sputter coat approximately 50 nm of gold or gold/palladium onto the surface of the deposit.

1.5.3.3.   SEM calibration and operation

1.5.3.3.1.   Calibration

The SEM calibration should be checked at least once a week (ideally once a day) using a certified calibration grid. The calibration should be checked against a certified standard and if the measured value (SEM) is not within ± 2 % of the certified value, then the SEM calibration must be adjusted and re-checked.

The SEM should be capable of resolving at least a minimum visible diameter of 0,2 μm, using a real sample matrix, at a magnification of × 2 000 .

1.5.3.3.2.   Operation

The SEM should be operated at 10 000 magnification ( 15 ) using conditions that give good resolution with an acceptable image at slow scan rates of, for example, 5 seconds per frame. Although the operational requirements of different SEMs may vary, generally to obtain the best visibility and resolution, with relatively low atomic weight materials, accelerating voltages of 5-10 keV should be used with a small spot size setting and short working distance. As a linear traverse is being conducted, then a 0° tilt should be used to minimise re-focussing or, if the SEM has a eucentric stage, the eucentric working distance should be used. Lower magnification may be used if the material does not contain small (diameter) fibres and the fibre diameters are large (> 5 μm).

1.5.3.4.   Sizing

1.5.3.4.1.   Low magnification examination to assess the sample

Initially the sample should be examined at low magnification to look for evidence of clumping of large fibres and to assess the fibre density. In the event of excessive clumping it is recommended that a new sample is prepared.

For statistical accuracy it is necessary to measure a minimum number of fibres and high fibre density may seem desirable as examining empty fields is time consuming and does not contribute to the analysis. However, if the filter is overloaded, it becomes difficult to measure all the measurable fibres and, because small fibres may be obscured by larger ones, they may be missed.

Bias towards over estimating the LWGMD may result from fibre densities in excess of 150 fibres per millimetre of linear traverse. On the other hand, low fibre concentrations will increase the time of analysis and it is often cost effective to prepare a sample with a fibre density closer to the optimum than to persist with counts on low concentration filters. The optimum fibre density should give an average of about one or two countable fibre per fields of view at 5 000 magnification. Nevertheless the optimum density will depend on the size (diameter) of the fibres, so it is necessary that the operator uses some expert judgement in order to decide whether the fibre density is close to optimal or not.

1.5.3.4.2.   Length weighting of the fibre diameters

Only those fibres that touch (or cross) an (infinitely) thin line drawn on the screen of the SEM are counted. For this reason a horizontal (or vertical) line is drawn across the centre of the screen.

Alternatively a single point is placed at the centre of the screen and a continuous scan in one direction across the filter is initiated. Each fibre of aspect ratio grater than 3:1 touching or crossing this point has its diameter measured and recorded.

1.5.3.4.3.   Fibre sizing

It is recommended that a minimum of 300 fibres are measured. Each fibre is measured only once at the point of intersection with the line or point drawn on the image (or close to the point of intersection if the fibre edges are obscured). If fibres with non-uniform cross sections are encountered, a measurement representing the average diameter of the fibre should be used. Care should be taken in defining the edge and measuring the shortest distance between the fibre edges. Sizing may be done on line, or off-line on stored images or photographs. Semi-automated image measurement systems that download data directly into a spreadsheet are recommended, as they save time, eliminate transcription errors and calculations can be automated.

The ends of long fibres should be checked at low magnification to ensure that they do not curl back into the measurement field of view and are only measured once.

2.   DATA

2.1.   TREATMENT OF RESULTS

Fibre diameters do not usually have a normal distribution. However, by performing a log transformation it is possible to obtain a distribution that approximates to normal.

Calculate the arithmetic mean (mean lnD) and the standard deviation (SDlnD) of the log to base e values (lnD) of the n fibre diameters (D).



image

(1)

image

(2)

The standard deviation is divided by the square root of the number of measurements (n) to obtain the standard error (SElnD).



image

(3)

Subtract two times the standard error from the mean and calculate the exponential of this value (mean minus two standard errors) to give the geometric mean minus two geometric standard errors.



image

(4)

3.   REPORTING

TEST REPORT

The test report should include at least the following information:

 The value of LWGMD-2SE.

 Any deviations and particularly those which may have an effect on the precision or accuracy of the results with appropriate justifications.

4.   REFERENCES

1. B. Tylee SOP MF 240. Health and Safety Executive, February 1999.

2. G. Burdett and G. Revell. Development of a standard method to measure the length-weigthed geometric mean fibre diameter: Results of the Second inter-laboratory exchange. IR/L/MF/94/07. Project R42.75 HPD. Health and Safety Executive, Research and Laboratory Services Division, 1994.

▼M4

A.23.   PARTITION COEFFICIENT (1-OCTANOL/WATER): SLOW-STIRRING METHOD

INTRODUCTION

1. This Test Method is equivalent to OECD Test Guideline (TG) 123 (2006). 1-octanol/water partition coefficient (POW) values up to a log POW of 8,2 have been accurately determined by the slow-stirring method (1). Therefore it is a suitable experimental approach for the direct determination of POW of highly hydrophobic substances.

2. Other methods for the determination of the 1-octanol/water partition coefficient (POW) are the ‘shake-flask’ method (2), and the determination of the POW from reversed phase HPLC-retention behaviour (3). The ‘shake-flask’ method is prone to artifacts due to transfer of octanol micro-droplets into the aqueous phase. With increasing values of POW the presence of these droplets in the aqueous phase leads to an increasing overestimation of the concentration of the test substance in the water. Therefore, its use is limited to substances with log POW < 4. The second method relies on solid data of directly determined POW values to calibrate the relationship between HPLC-retention behaviour and measured values of POW. A draft OECD guideline was available for determining 1-octanol/water partition coefficients of ionisable substances (4) but shall no longer be used.

3. This Test Method has been developed in The Netherlands. The precision of the methods described here has been validated and optimized in a ring-test validation study in which 15 laboratories participated (5).

INITIAL CONSIDERATIONS

Significance and use

4. For inert organic substances highly significant relationships have been found between 1-octanol/water partition coefficients (POW) and their bioaccumulation in fish. Moreover, POW has been demonstrated to be correlated to fish toxicity as well as to sorption of chemicals to solids such as soils and sediments. An extensive overview of the relationships has been given in reference (6).

5. A wide variety of relationships between the 1-octanol/water partition coefficient and other substance properties of relevance to environmental toxicology and chemistry have been established. As a consequence, the 1-octanol/water partition coefficient has evolved as a key parameter in the assessment of the environmental risk of chemicals as well as in the prediction of fate of chemicals in the environment.

Scope

6. The slow-stirring experiment is thought to reduce the formation of micro-droplets from 1-octanol droplets in the water phase. As a consequence, overestimation of the aqueous concentration due to test substance molecules associated to such droplets does not occur. Therefore, the slow-stirring method is particularly suitable for the determination of POW for substances with expected log POW values of 5 and higher, for which the shake-flask method (2) is prone to yield erroneous results.

DEFINITION AND UNITS

7. The partition coefficient of a substance between water and a lipophilic solvent (1-octanol) characterizes the equilibrium distribution of the chemical between the two phases. The partition coefficient between water and 1-octanol (POW) is defined as the ratio of the equilibrium concentrations of the test substance in 1-octanol saturated with water (CO) and water saturated with 1-octanol (CW).

image

As a ratio of concentrations it is dimensionless. Most frequently it is given as the logarithm to the base 10 (log POW). POW is temperature dependent and reported data should include the temperature of the measurement.

PRINCIPLE OF THE METHOD

8. In order to determine the partitioning coefficient, water, 1-octanol, and the test substance are equilibrated with each other at constant temperature. Then the concentrations of the test substance in the two phases are determined.

9. The experimental difficulties associated with the formation of micro-droplets during the shake-flask experiment can be reduced in the slow-stirring experiment proposed here. In the slow-stirring experiment, water, 1-octanol and the test substance are equilibrated in a thermostated stirred reactor. Exchange between the phases is accelerated by stirring. The stirring introduces limited turbulence which enhances the exchange between 1-octanol and water without micro-droplets being formed (1).

APPLICABILITY OF THE TEST

10. Since the presence of substances other than the test substance might influence the activity coefficient of the test substance, the test substance should be tested as a pure substance. The highest purity commercially available should be employed for the 1-octanol/water partition experiment.

11. The present method applies to pure substances that do not dissociate or associate and that do not display significant interfacial activity. It can be applied to determine the 1-octanol/water partition ratio of such substances and of mixtures. When the method is used for mixtures, the 1-octanol/water partition ratios determined are conditional and depend on the chemical composition of the mixture tested and on the electrolyte composition employed as aqueous phase. Provided additional steps are taken, the method is also applicable to dissociating or associating compounds (paragraph 12).

12. Due to the multiple equilibria in water and 1-octanol involved in the 1-octanol/water partitioning of dissociating substances such as organic acids and phenols, organic bases, and organometallic substances, the 1-octanol/water partition ratio is a conditional constant strongly dependent on electrolyte composition (7)(8). Determination of the 1-octanol/water partition ratio therefore requires that pH and electrolyte composition be controlled in the experiment and reported. Expert judgement has to be employed in the evaluation of these partition ratios. Using the value of dissociation constant(s), suitable pH-values need to be selected, such that a partitioning ratio is determined for each ionization state. Non-complexing buffers must be used when testing organometallic compounds (8). Taking the existing knowledge on the aqueous chemistry (complexation constants, dissociation constants) into account, the experimental conditions should be chosen in such a manner that the speciation of the test substance in the aqueous phase can be estimated. The ionic strength should be identical in all experiments by employing a background electrolyte.

13. Difficulties in the test may arise in conducting the test for substances with low water solubility or high POW, due to the fact that the concentrations in the water become very low such that their accurate determination is difficult. This Test Method provides guidance on how to deal with this problem.

INFORMATION ON THE TEST SUBSTANCE

14. Chemical reagents should be of analytical grade or of higher purity. The use of non-labelled test substances with known chemical composition and preferably at least 99 % purity, or of radiolabelled test substances with known chemical composition and radiochemical purity, is recommended. In the case of short half-life tracers, decay corrections should be applied. In the case of radiolabelled test substances, a chemical specific analytical method should be employed to ensure that the measured radioactivity is directly related to the test substance.

15. An estimate of log POW may be obtained by using commercially available software for estimation of log POW, or by using the ratio of the solubilities in both solvents.

16. Before carrying out a slow-stirring experiment for determination of POW, the following information on the test substance should be available:

(a) structural formula

(b) suitable analytical methods for determination of the concentration of the substance in water and 1-octanol

(c) dissociation constant(s) of ionisable substances (OECD Guideline 112 (9))

(d) aqueous solubility (10)

(e) abiotic hydrolysis (11)

(f) ready biodegradability (12)

(g) vapour pressure (13).

DESCRIPTION OF THE METHOD

Equipment and apparatus

17. Standard laboratory equipment is required, in particular, the following:

 magnetic stirrers and Teflon coated magnetic stir bars are employed to stir the water phase;

 analytical instrumentation, suitable for determination of the concentration of the test substance at the expected concentrations;

 stirring-vessel with a tap at the bottom. Dependent on the estimate of log POW and the Limit of Detection (LOD) of the test compound, the use of a reaction vessel of the same geometry larger than one litre has to be considered, so that a sufficient volume of water can be obtained for chemical extraction and analysis. This will result in higher concentrations in the water extract and thus a more reliable analytical determination. A table giving estimates of the minimum volume needed, the LOD of the compound, its estimated log POW and its water solubility is given in Appendix 1. The table is based on the relationship between log POW and the ratio between the solubilities in octanol and water, as presented by Pinsuwan et al. (14):

  image

 where

  image (in molarity);

 and the relationship given by Lyman (15) for predicting water solubility. Water solubilities calculated with the equation given in Appendix 1 must be seen as a first estimate. It should be noted that the user is free to generate an estimate of water solubility by means of any relationship that is considered to better represent the relationship between hydrophobicity and solubility. For solid compounds, inclusion of melting point in the prediction of solubility is for instance recommended. In case a modified equation is used, it should be ascertained that the equation for calculation of solubility in octanol is still valid. A schematic drawing of a glass-jacketed stirring-vessel with a volume of ca. one litre is given in Appendix 2. The proportions of the vessel shown in Appendix 2 have proven favourable and should be maintained when apparatus of a different size is used;

 a means for keeping the temperature constant during the slow-stirring experiment is essential.

18. Vessels should be made from inert material such that adsorption to vessel surfaces is negligible.

Preparation of the test solutions

19. The POW determination should be carried out with the highest purity 1-octanol that is commercially available (at least + 99 %). Purification of 1-octanol by extraction with acid, base and water and subsequent drying is recommended. In addition, distillation can be used to purify 1-octanol. Purified 1-octanol is to be used to prepare standard solutions of the test substances. Water to be used in the POW determination should be glass or quartz distilled, or obtained from a purification system, or HPLC-grade water may be used. Filtration through a 0,22 μm filter is required for distilled water, and blanks should be included to check that no impurities are in the concentrated extracts that may interfere with the test substance. If a glass fibre filter is used, it should be cleaned by baking for at least three hours at 400 °C.

20. Both solvents are mutually saturated prior to the experiment by equilibrating them in a sufficiently large vessel. This is accomplished by slow-stirring the two-phase system for two days.

21. An appropriate concentration of test substance is selected and dissolved in 1-octanol (saturated with water). The 1-octanol/water partition coefficient needs to be determined in dilute solutions in 1-octanol and water. Therefore the concentration of the test substance should not exceed 70 % of its solubility with a maximum concentration of 0,1 M in either phase (1). The 1-octanol solutions used for the experiment must be devoid of suspended solid test substance.

22. The appropriate amount of test substance is dissolved in 1-octanol (saturated with water). If the estimate of log POW exceeds five, care has to be taken that the 1-octanol solutions used for the experiment are devoid of suspended solid test substance. To that end, the following procedure for chemicals with an estimated value of log POW > 5 is followed:

 the test substance is dissolved in 1-octanol (saturated with water);

 the solution is given sufficient time for the suspended solid substance to settle out. During the settling period, the concentration of the test substance is monitored;

 after the measured concentrations in the 1-octanol-solution have attained stable values, the stock solution is diluted with an appropriate volume of 1-octanol;

 the concentration of the diluted stock solution is measured. If the measured concentration is consistent with the dilution, the diluted stock solution can be employed in the slow-stirring experiment.

Extraction and analysis of samples

23. A validated analytical method should be used for the assay of test substance. The investigators have to provide evidence that the concentrations in the water saturated 1-octanol as well as in the 1-octanol saturated water phase during the experiment are above the method limit of quantification of the analytical procedures employed. Analytical recoveries of the test substance from the water phase and from the 1-octanol phase need to be established prior to the experiment in those cases for which extraction methods are necessary. The analytical signal needs to be corrected for blanks and care should be taken that no carry-over of analyte from one sample to another can occur.

24. Extraction of the water phase with an organic solvent and preconcentration of extract are likely to be required prior to analysis, due to rather low concentrations of hydrophobic test substances in the water phase. For the same reason it is necessary to reduce eventual blank concentrations. To that end, it is necessary to employ high purity solvents, preferably solvents for residue analysis. Moreover, working with carefully pre-cleaned (e.g. solvent washing or baking at elevated temperature) glassware can help to avoid cross-contamination.

25. An estimate of log POW may be obtained from an estimation program or by expert judgment. If the value is higher than six then blank corrections and analyte carry-over need to be monitored closely. Similarly, if the estimate of log POW exceeds six, the use of a surrogate standard for recovery correction is mandatory, so that high preconcentration factors can be reached. A number of software programs for the estimation of log POW are commercially available ( 16 ), e.g. Clog P (16), KOWWIN (17), ProLogP (18) and ACD log P (19). Descriptions of the estimation approaches can be found in references (20-22).

26. The limits of quantification (LOQ) for determination of the test substance in 1-octanol and water are established using accepted methods. As a rule of thumb, the method limit of quantification can be determined as the concentration in water or 1-octanol that produces a signal to noise ratio of ten. A suitable extraction and pre-concentration method should be selected and analytical recoveries should also be specified. A suitable pre-concentration factor is selected in order to obtain a signal of the required size upon analytical determination.

27. On the basis of the parameters of the analytical method and the expected concentrations, an approximate sample size required for an accurate determination of the compound concentration is determined. The use of water samples that are too small to obtain a sufficient analytical signal should be avoided. Also, the use of excessively large water samples should be avoided, since otherwise there might be too little water left for the minimum number of analyses required (n = 5). In Appendix 1, the minimum sample volume is indicated as a function of the vessel volume, the LOD of the test substance and the solubility of the test substance.

28. Quantification of the test substances occurs by comparison with calibration curves of the respective compound. The concentrations in the samples analysed must be bracketed by concentrations of standards.

29. For test substances with a log POW estimate higher than six a surrogate standard has to be spiked to the water sample prior to extraction in order to register losses occurring during extraction and pre-concentration of the water samples. For accurate recovery correction, the surrogates must have properties that are very close to, or identical with, those of the test substance. Preferably, (stable) isotopically-labelled analogues of the substances of interest (for example, perdeuterated or 13C-labelled) are used for this purpose. If the use of labelled stable isotopes, i.e. 13C or 2H, is not possible it should be demonstrated from reliable data in the LITERATURE that the physical-chemical properties of the surrogate are very close to those of the test substance. During liquid-liquid extraction of the water phase emulsions can form. They can be reduced by addition of salt and allowing the emulsion to settle overnight. Methods used for extracting and pre-concentrating the samples need to be reported.

30. Samples withdrawn from the 1-octanol phase may, if necessary, be diluted with a suitable solvent prior to analysis. Moreover, the use of surrogate standards for recovery correction is recommended for substances for which the recovery experiments demonstrated a high degree of variation in the recovery experiments (relative standard deviation > 10 %).

31. The details of the analytical method need to be reported. This includes the method of extraction, pre-concentration and dilution factors, instrument parameters, calibration routine, calibration range, analytical recovery of the test substance from water, addition of surrogate standards for recovery correction, blank values, detection limits and limits of quantification.

Performance of the Test

Optimal 1-octanol/water volume ratios

32. When choosing the water and 1-octanol volumes, the LOQ in 1-octanol and water, the pre-concentration factors applied to the water samples, the volumes sampled in 1-octanol and water, and the expected concentrations should be considered. For experimental reasons, the volume of 1-octanol in the slow-stirring system should be chosen such that the 1-octanol layer is sufficiently thick (> 0,5 cm) in order to allow for sampling of the 1-octanol phase without disturbing it.

33. Typical phase ratios used for the determinations of compounds with log POW of 4,5 and higher are 20 to 50 ml of 1-octanol and 950 to 980 ml of water in a one litre vessel.

Test conditions

34. During the test the reaction vessel is thermostated to reduce temperature variation to below 1 °C. The assay should be performed at 25 °C.

35. The experimental system should be protected from daylight by either performing the experiment in a dark room or by covering the reaction vessel with aluminium foil.

36. The experiment should be performed in a dust-free (as far as possible) environment.

37. The 1-octanol-water system is stirred until equilibrium is attained. In a pilot experiment the length of the equilibration period is assessed by performing a slow-stirring experiment and sampling water and 1-octanol periodically. The sampling time points should be interspersed by a minimum period of five hours.

38. Each POW determination has to be performed employing at least three independent slow-stirring experiments.

Determination of the equilibration time

39. It is assumed that the equilibrium is achieved when a regression of the 1-octanol/water concentration ratio against time over a time span of four time points yields a slope that is not significantly different from zero at a p-level of 0,05. The minimum equilibration time is one day before sampling can be started. As a rule of thumb, sampling of substances with a log POW estimate of less than five can take place during days two and three. The equilibration might have to be extended for more hydrophobic compounds. For a compound with log POW of 8,23 (decachlorobiphenyl) 144 hours were sufficient for equilibration. Equilibrium is assessed by means of repeated sampling of a single vessel.

Starting the experiment

40. At the start of the experiment the reaction vessel is filled with 1-octanol-saturated water. Sufficient time should be allowed to reach the thermostated temperature.

41. The desired amount of test substance (dissolved in the required volume of 1-octanol saturated with water) is carefully added to the reaction vessel. This is a crucial step in the experiment, since turbulent mixing of the two phases has to be avoided. To that end, the 1-octanol phase can be pipetted slowly against the wall of the experimental vessel, close to the water surface. It will subsequently flow along the glass wall and form a film above the water phase. The decantation of 1-octanol directly into the flask should always be avoided; drops of 1-octanol should not be allowed to fall directly into the water.

42. After starting the stirring, the stirring rate should be increased slowly. If the stirring motors cannot be appropriately adjusted the use of a transformer should be considered. The stirring rate should be adjusted so that a vortex at the interface between water and 1-octanol of 0,5 to maximally 2,5 cm depth is created. The stirring rate should be reduced if the vortex depth of 2,5 cm is exceeded; otherwise micro-droplets may be formed from 1-octanol droplets in the water phase, leading to an overestimation of the concentration of the test substance in the water. The maximum stirring rate of 2,5 cm is recommended on the basis of the findings in the ring-test validation study (5). It is a compromise between achieving a rapid rate of equilibration, while limiting the formation of 1-octanol micro-droplets.

Sampling and Sample Treatment

43. The stirrer should be turned off prior to sampling and the liquids should be allowed to stop moving. After sampling is completed, the stirrer is started again slowly, as described above, and then the stirring rate is increased gradually.

44. The water phase is sampled from a stopcock at the bottom of the reaction vessel. Always discard the dead volume of water contained in the taps (approximately 5 ml in the vessel shown in the Appendix 2). The water in the taps is not stirred and therefore not in equilibrium with the bulk. Note the volume of the water samples, and make sure that the amount of test substance present in the discarded water is taken into account when setting up a mass balance. Evaporative losses should be minimized by allowing the water to flow quiescently into the separatory funnel, such that there is no disturbance of the water/1-octanol layer.

45. 1-Octanol samples are obtained by withdrawing a small aliquot (ca. 100 μl) from the 1-octanol layer with a 100 microlitre all glass-metal syringe. Care should be taken not to disturb the boundary. The volume of the sampled liquid is recorded. A small aliquot is sufficient, since the 1-octanol sample will be diluted.

46. Unnecessary sample transfer steps should be avoided. To that end the sample volume should be determined gravimetrically. In case of water samples this can be achieved by collecting the water sample in a separatory funnel that contains already the required volume of solvent.

DATA AND REPORTING

47. According to the present Test Method, POW is determined by performing three slow-stirring experiments (three experimental units) with the compound under investigation employing identical conditions. The regression used to demonstrate attainment of equilibrium should be based on the results of at least four determinations of CO/CW at consecutive time points. This allows for calculating variance as a measure of the uncertainty of the average value obtained by each experimental unit.

48. The POW can be characterized by the variance in the data obtained for each experimental unit. This information is employed to calculate the POW as the weighted average of the results of the individual experimental units. To do so, the inverse of the variance of the results of the experimental units is employed as weight. As a result, data with a large variation (expressed as the variance) and thus with lower reliability have less influence on the result than data with a low variance.

49. Analogously, the weighted standard deviation is calculated. It characterizes the repeatability of the POW measurement. A low value of the weighted standard deviation indicates that the POW determination was very repeatable within one laboratory. The formal statistical treatment of the data is outlined below.

Treatment of the results

Demonstration of attainment of equilibrium

50. The logarithm of the ratio of the concentration of the test substance in 1-octanol and water (log (CO/Cw)) is calculated for each sampling time. Achievement of chemical equilibrium is demonstrated by plotting this ratio against time. A plateau in this plot that is based on at least four consecutive time points indicates that equilibrium has been attained, and that the compound is truly dissolved in 1-octanol. If not, the test needs to be continued until four successive time points yield a slope that is not significantly different from 0 at a p-level of 0,05, indicating that log Co/Cw is independent of time.

Log POW-calculation

51. The value of log POW of the experimental unit is calculated as the weighted average value of log Co/Cw for the part of the curve of log Co/Cw vs. time, for which equilibrium has been demonstrated. The weighted average is calculated by weighting the data with the inverse of the variance so that the influence of the data on the final result is inversely proportional to the uncertainty in the data.

Average log POW

52. The average value of log POW of different experimental units is calculated as the average of the results of the individual experimental units weighted with their respective variances.

The calculation is performed as follows:

image

where:

log POW,i

=

the log POW value of the individual experimental unit i;

log POW,Av

=

the weighted average value of the individual log POW determinations;

wi

=

the statistical weight assigned to the log POW value of the experimental unit i.

The reciprocal of the variance of log POW,i is employed as wi ( image )

53. The error of the average of log POW is estimated as the repeatability of log Co/Cw determined during the equilibrium phase in the individual experimental units. It is expressed as the weighted standard deviation of log POW,Avlog Pow,Av) which in turn is a measure of the error associated with log POW,Av. The weighted standard deviation can be computed from the weighted variance (varlog Pow,Av) as follows:

image

image

The symbol n stands for the number of experimental units.

Test Report

54. The test report should include the following information:

Test substance:

 common name, chemical name, CAS number, structural formula (indicating position of label when radiolabelled substance is used) and relevant physical-chemical properties (see paragraph 17)

 purity (impurities) of test substance

 label purity of labelled chemicals and molar activity (where appropriate)

 the preliminary estimate of log Pow, as well as the method used to derive the value.

Test conditions:

 dates of the performance of the studies

 temperature during the experiment

 volumes of 1-octanol and water at the beginning of the test

 volumes of withdrawn 1-octanol and water samples

 volumes of 1-octanol and water remaining in the test vessels

 description of the test vessels and stirring conditions (geometry of the stirring bar and of the test vessel, vortex height in mm, and when available: stirring rate) used

 analytical methods used to determine the test substance and the method limit of quantification

 sampling times

 the aqueous phase pH and the buffers used, when pH is adjusted for ionizable molecules

 number of replicates.

Results:

 repeatability and sensitivity of the analytical methods used

 determined concentrations of the test substance in 1-octanol and water as a function of time

 demonstration of mass balance

 temperature and standard deviation or the range of temperature during the experiment

 the regression of concentration ratio against time

 the average value log Pow,Av and its standard error

 discussion and interpretation of the results

 examples of raw data figures of representative analysis (all raw data have to be stored in accordance with GLP standards), including recoveries of surrogates, and the number of levels used in the calibration (along with the criteria for the correlation coefficient of the calibration curve), and results of quality assurance/quality control (QA/QC)

 when available: validation report of the assay procedure (to be indicated among references).

LITERATURE:

(1) De Bruijn JHM, Busser F, Seinen W, Hermens J. (1989). Determination of octanol/water partition coefficients with the ‘slow-stirring’ method. Environ. Toxicol. Chem. 8: 499-512.

(2) Chapter A.8 of this Annex, Partition Coefficient.

(3) Chapter A.8 of this Annex, Partition Coefficient.

(4) OECD (2000). OECD Draft Guideline for the Testing of Chemicals: 122 Partition Coefficient (n-Octanol/Water): pH-Metric Method for Ionisable Substances. Paris.

(5) Tolls J (2002). Partition Coefficient 1-Octanol/Water (Pow) Slow-Stirring Method for Highly Hydrophobic Chemicals, Validation Report. RIVM contract-Nrs 602730 M/602700/01.

(6) Boethling RS, Mackay D (eds.) (2000). Handbook of property estimation methods for chemicals. Lewis Publishers Boca Raton, FL, USA.

(7) Schwarzenbach RP, Gschwend PM, Imboden DM (1993). Environmental Organic Chemistry. Wiley, New York, NY.

(8) Arnold CG, Widenhaupt A, David MM, Müller SR, Haderlein SB, Schwarzenbach RP (1997). Aqueous speciation and 1-octanol-water partitioning of tributyl- and triphenyltin: effect of pH and ion composition. Environ. Sci. Technol. 31: 2596-2602.

(9) OECD (1981) OECD Guidelines for the Testing of Chemicals: 112 Dissociation Constants in Water. Paris.

(10) Chapter A.6 of this Annex, Water Solubility.

(11) Chapter C.7 of this Annex, Degradation – Abiotic Degradation Hydrolysis as a Function of pH.

(12) Chapter C.4 — Part II – VII (Method A to F) of this Annex, Determination of ‘Ready’ Biodegradability.

(13) Chapter A.4 of this Annex, Vapour Pressure.

(14) Pinsuwan S, Li A and Yalkowsky S.H. (1995). Correlation of octanol/water solubility ratios and partition coefficients, J. Chem. Eng. Data. 40: 623-626.

(15) Lyman WJ (1990). Solubility in water. In: Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds, Lyman WJ, Reehl WF, Rosenblatt DH, Eds. American Chemical Society, Washington, DC, 2-1 to 2-52.

(16) Leo A, Weininger D (1989). Medchem Software Manual. Daylight Chemical Information Systems, Irvine, CA.

(17) Meylan W (1993). SRC-LOGKOW for Windows. SRC, Syracuse, N.Y.

(18) Compudrug L (1992). ProLogP. Compudrug, Ltd, Budapest.

(19) ACD. ACD logP; Advanced Chemistry Development: Toronto, Ontario M5H 3V9, Canada, 2001.

(20) Lyman WJ (1990). Octanol/water partition coefficient. In Lyman WJ, Reehl WF, Rosenblatt DH, eds, Handbook of chemical property estimation, American Chemical Society, Washington, D.C.

(21) Rekker RF, de Kort HM (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. Chim. Ther. 14: 479-488.

(22) Jübermann O (1958). Houben-Weyl, ed, Methoden der Organischen Chemie: 386-390.

Appendix 1

Spreadsheet for computation of minimum volumes of water required for detection of test substances of different log POW values in aqueous phase

Assumptions:

 Maximum volume of individual aliquots = 10 % of total volume; 5 aliquots = 50 % of total volume.

  image . In case of lower concentrations, larger volumes would be required.

 Volume used for LOD determination = 100 ml.

 log Pow vs. log Sw and log Pow vs. SR (Soct/Sw) are reasonable representations of relationships for test substances.

Estimation of Sw



log Pow

Equation

log Sw

Sw (mg/l)

4

image

0,496

3,133E+00

4,5

image

0,035

1,084E+00

5

image

–0,426

3,750E-01

5,5

image

–0,887

1,297E-01

6

image

–1,348

4,487E-02

6,5

image

––1,809

1,552E-02

7

image

–2,270

5,370E-03

7,5

image

–2,731

1,858E-03

8

image

–3,192

6,427E-04

Estimation of Soct



log Pow

Equation

Soct (mg/l)

4

image

3,763E+04

4,5

image

4,816E+04

5

image

6,165E+04

5,5

image

7,890E+04

6

image

1,010E+05

6,5

image

1,293E+05

7

image

1,654E+05

7,5

image

2,117E+05

8

image

2,710E+05



Total Mass test substance

(mg)

Massoct/Masswater

MassH2O

(mg)

ConcH2O

(mg/l)

Massoct

(mg)

Concoct

(mg/l)

1 319

526

2,5017

2,6333

1 317

26 333

1 686

1 664

1,0127

1,0660

1 685

33 709

2 158

5 263

0,4099

0,4315

2 157

43 149

2 762

16 644

0,1659

0,1747

2 762

55 230

3 535

52 632

0,0672

0,0707

3 535

70 691

4 524

1664 36

0,0272

0,0286

4 524

90 480

5 790

5263 16

0,0110

0,0116

5 790

115 807

7 411

1 664 357

0,0045

0,0047

7 411

148 223

9 486

5 263 158

0,0018

0,0019

9 486

189 713

Computation of volumes



Minimum volume required for H2O phase at each LOD concentration

log Kow

LOD (micrograms/l)→

0,001

0,01

0,10

1,00

10

4

 

0,04

0,38

3,80

38

380

4,5

 

0,09

0,94

9,38

94

938

5

 

0,23

2,32

23,18

232

2 318

5,5

 

0,57

5,73

57,26

573

5 726

6

 

1,41

14,15

141

1 415

14 146

6,5

 

3,50

34,95

350

3 495

34 950

7

 

8,64

86,35

864

8 635

86 351

7,5

 

21,33

213

2 133

21 335

213 346

8

 

52,71

527

5 271

52 711

527 111

Volume used for LOD (l)

0,1

 

 

 

 

 

Key to Computations

Represents < 10 % of total volume of aqueous phase, 1 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 2 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 5 litre equilibration vessel.

Represents < 10 % of total volume of aqueous phase, 10 litre equilibration vessel.

Exceeds 10 % of even the 10 liter equilibration vessel.



Overview of volumes required, as a function of water solubility and Log Pow

Minimum volume required for H2O phase at each LOD concentration (ml)

log Pow

Sw (mg/l)

LOD (micrograms/l)→

0,001

0,01

0,10

1,00

10

4

10

 

0,01

0,12

1,19

11,90

118,99

 

5

 

0,02

0,24

2,38

23,80

237,97

 

3

 

0,04

0,40

3,97

39,66

396,62

 

1

 

0,12

1,19

11,90

118,99

1 189,86

4,5

5

 

0,02

0,20

2,03

20,34

203,37

 

2

 

0,05

0,51

5,08

50,84

508,42

 

1

 

0,10

1,02

10,17

101,68

1 016,83

 

0,5

 

0,20

2,03

20,34

203,37

2 033,67

5

1

 

0,09

0,87

8,69

86,90

869,01

 

0,5

 

0,17

1,74

17,38

173,80

1 738,02

 

0,375

 

0,23

2,32

23,18

231,75

2 317,53

 

0,2

 

0,43

4,35

43,45

434,51

4 345,05

5,5

0,4

 

0,19

1,86

18,57

185,68

1 856,79

 

0,2

 

0,37

3,71

37,14

371,36

3 713,59

 

0,1

 

0,74

7,43

74,27

742,72

7 427,17

 

0,05

 

1,49

14,85

148,54

1 485,43

14 854,35

6

0,1

 

0,63

6,35

63,48

634,80

6 347,95

 

0,05

 

1,27

12,70

126,96

1 269,59

12 695,91

 

0,025

 

2,54

25,39

253,92

2 539,18

25 391,82

 

0,0125

 

5,08

50,78

507,84

5 078,36

50 783,64

6,5

0,025

 

2,17

21,70

217,02

2 170,25

21 702,46

 

0,0125

 

4,34

43,40

434,05

4 340,49

43 404,93

 

0,006

 

9,04

90,43

904,27

9 042,69

90 426,93

 

0,003

 

18,09

180,85

1 808,54

18 085,39

180 853,86

7

0,006

 

7,73

77,29

772,89

7 728,85

77 288,50

 

0,003

 

15,46

154,58

1 545,77

15 457,70

154 577,01

 

0,0015

 

23,19

231,87

2 318,66

23 186,55

231 865,51

 

0,001

 

46,37

463,73

4 637,31

46 373,10

463 731,03

7,5

0,002

 

19,82

198,18

1 981,77

19 817,73

198 177,33

 

0,001

 

39,64

396,35

3 963,55

39 635,47

396 354,66

 

0,0005

 

79,27

792,71

7 927,09

79 270,93

792 709,32

 

0,00025

 

158,54

1 585,42

15 854,19

158 541,86

1 585 418,63

8

0,001

 

33,88

338,77

3 387,68

33 876,77

338 767,72

 

0,0005

 

67,75

677,54

6 775,35

67 753,54

677 535,44

 

0,00025

 

135,51

1 355,07

13 550,71

135 507,09

1 355 070,89

 

0,000125

 

271,01

2 710,14

27 101,42

271 014,18

2 710 141,77

Volume used for LOD (l)

0,1

 

 

 

 

 

Appendix 2

An example of glass-jacketed test vessel for the slow-stirring experiment for determination of POW

image

▼M6

A.24.   PARTITION COEFFICIENT (N-OCTANOL/WATER), HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC) METHOD

INTRODUCTION

This test method is equivalent to OECD test guideline (TG) 117 (2004)

1. The partition coefficient (P) is defined as the ratio of the equilibrium concentrations of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case of n-octanol and water,

image

The partition coefficient being the quotient of two concentrations, is dimensionless and is usually given in the form of its logarithm to base ten.

2. Pow is a key parameter in studies of the environmental fate of chemical substances. A highly-significant relationship between the Pow of non-ionised form of substances and their bioaccumulation in fish has been shown. It has also been shown that Pow is a useful parameter in the prediction of adsorption on soil and sediments and for establishing quantitative structure-activity relationships for a wide range of biological effects.

3. The original proposal for this test method was based on an article by C.V. Eadsforth and P. Moser (1). The development of the test method and an OECD inter-laboratory comparison test were coordinated by the Umweltbundesamt of the Federal Republic of Germany during 1986 (2).

INITIAL CONSIDERATIONS

4. log Pow values in the range – 2 to 4 (occasionally up to 5 and more) ( 17 ) can be experimentally determined by the Shake-Flask method (Chapter A.8 of this Annex, OECD Test Guideline 107). The HPLC method covers log Pow in the range of 0 to 6 (1)(2)(3)(4)(5). This method may require an estimation of Pow to assign suitable reference substances and support any conclusions drawn from the data generated by the test. Calculation methods are briefly discussed in the Appendix to this test method. The HPLC operation mode is isocratic.

5. The Pow values depend on the environmental conditions such as temperature, pH, ionic strength etc, and these should be defined in the experiment for the correct interpretation of Pow data. For ionisable substances, another method (e.g. draft OECD guideline on pH metric method for ionised substances (6)) may become available and could be used as an alternative method. Although this draft OECD guideline may appropriate be suitable to determine Pow for those ionisable substances, in some cases it is more appropriate to use the HPLC method at an environmentally relevant pH (see paragraph 9).

PRINCIPLE OF THE METHOD

6. Reverse phase HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica.

7. A chemical injected on such a column partitions between the mobile solvent phase and the hydrocarbon stationary phase as it is transported along the column by the mobile phase. The substances are retained in proportion to their hydrocarbon-water partition coefficient, with hydrophilic substances eluted first and lipophilic substances last. The retention time is described by the capacity factor k given by the expression:

image

where tR is the retention time of the test substance, and t0 is the dead-time, i.e. the average time a solvent molecule needs to pass the column. Quantitative analytical methods are not required and only the determination of retention times is necessary.

8. The octanol/water partition coefficient of a test substance can be computed by experimentally determining its capacity factor k and then inputting k into the following equation:

image

where

a, b

=

linear regression coefficients.

The equation above can be obtained by linearly regressing the log of octanol/water partition coefficients of reference substances against the log of capacity factors of the reference substances.

9. Reverse phase HPLC method enables partition coefficients to be estimated in the log Pow range between 0 and 6, but can be expanded to cover the log Pow range between 6 and 10 in exceptional cases. This may require that the mobile phase is modified (3). The method is not applicable to strong acids and bases, metal complexes, substances which react with the eluent, or surface-active agents. Measurements can be performed on ionisable substances in their non-ionised form (free acid or free base) only by using an appropriate buffer with a pH below the pKa for a free acid or above the pKa for a free base. Alternatively, the pH-metric method for the testing of ionisable substances (6) may become available and could be used as an alternative method (6). If the log Pow value is determined for the use in environmental hazard classification or in environmental risk assessment, the test should be performed in the pH range relevant for the natural environment, i.e. in the pH range of 5,0 - 9.

10. In some cases impurities can make the interpretation of the results difficult due to uncertainty in peak assignments. For mixtures which result in an unresolved band, upper and lower limits of log Pow, and the area % of each log Pow peak should be reported. For mixtures which are a group of homologues, the weighted average log Pow should also be stated (7), calculated based on the single Pow values and the corresponding area % values (8). All peaks that contribute an area of 5 % or more to the total peak area should be taken into consideration in the calculation (9):

image

The weighed average log Pow is valid only for substances or mixtures (e.g. tall oils) consisting of homologues (e.g. series of alkanes). Mixtures can be measured with meaningful results, provided that the analytical detector used has the same sensitivity towards all the substances in the mixture and that they can be adequately resolved.

INFORMATION ON THE TEST SUBSTANCE

11. The dissociation constant, structural formula, and solubility in the mobile phase should be known before the method is used. In addition, information on hydrolysis would be helpful.

QUALITY CRITERIA

12. In order to increase the confidence in the measurement, duplicate determinations must be made.

 Repeatability: The value of log Pow derived from repeated measurements made under identical conditions and using the same set of reference substances should fall within a range of ± 0,1 log units.

 Reproducibility: If the measurements are repeated with a different set of reference substances, results may differ. Typically, the correlation coefficient R for the relationship between log k and log Pow for a set of test substances is around 0,9, corresponding to an octanol/water partition coefficient of log Pow ± 0,5 log units.

13. The inter-laboratory comparison test has shown that with the HPLC method log Pow values can be obtained to within ± 0,5 units of the Shake-Flask values (2). Other comparisons can be found in the literature (4)(5)(10)(11)(12). Correlation graphs based on structurally related reference substances give the most accurate results (13).

REFERENCE SUBSTANCES

14. In order to correlate the measured capacity factor k of a substance with its Pow, a calibration graph using at least 6 points has to be established (see paragraph 24). It is up to the user to select the appropriate reference substances. The reference substances should normally have log Pow values which encompass the log Pow of the test substance, i.e. at least one reference substance should have a Pow above that of the test substance, and another a Pow below that of the test substance. Extrapolation should only be used in exceptional cases. It is preferable that these reference substances should be structurally related to the test substance. log Pow values of the reference substances used for the calibration should be based on reliable experimental data. However, for substances with high log Pow (normally more than 4), calculated values may be used unless reliable experimental data are available. If extrapolated values are used a limit value should be quoted.

15. Extensive lists of log Pow values for many groups of chemicals are available (14)(15). If data on the partition coefficients of structurally related substances are not available, a more general calibration, established with other reference substances, may be used. Recommended reference substances and their Pow values are listed in Table 1. For ionisable substances the values given apply to the non-ionised form. The values were checked for plausibility and quality during the inter-laboratory comparison test.



Table 1

Recommended reference substances

 

CAS Number

Reference substance

log Pow

pKa

1

78-93-3

2-Butanone

(Methylethylketone)

0,3

 

2

1122-54-9

4-Acetylpyridine

0,5

 

3

62-53-3

Aniline

0,9

 

4

103-84-4

Acetanilide

1,0

 

5

100-51-6

Benzyl alcohol

1,1

 

6

150-76-5

4-Methoxyphenol

1,3

pKa = 10,26

7

122-59-8

Phenoxyacetic acid

1,4

pKa = 3,12

8

108-95-2

Phenol

1,5

pKa = 9,92

9

51-28-5

2,4-Dinitrophenol

1,5

pKa = 3,96

10

100-47-0

Benzonitrile

1,6

 

11

140-29-4

Phenylacetonitrile

1,6

 

12

589-18-4

4-Methylbenzyl alcohol

1,6

 

13

98-86-2

Acetophenone

1,7

 

14

88-75-5

2-Nitrophenol

1,8

pKa = 7,17

15

121-92-6

3-Nitrobenzoic acid

1,8

pKa = 3,47

16

106-47-8

4-Chloroaniline

1,8

pKa = 4,15

17

98-95-3

Nitrobenzene

1,9

 

18

104-54-1

Cinnamyl alcohol

(Cinnamic alcohol)

1,9

 

19

65-85-0

Benzoic acid

1,9

pKa = 4,19

20

106-44-5

p-Cresol

1,9

pKa = 10,17

21

140-10-3

(trans)

Cinnamic acid

2,1

pKa = 3,89 (cis)

4,44 (trans)

22

100-66-3

Anisole

2,1

 

23

93-58-3

Methyl benzoate

2,1

 

24

71-43-2

Benzene

2,1

 

25

99-04-7

3-Methylbenzoic acid

2,4

pKa = 4,27

26

106-48-9

4-Chlorophenol

2,4

pKa = 9,1

27

79-01-6

Trichloroethylene

2,4

 

28

1912-24-9

Atrazine

2,6

 

29

93-89-0

Ethyl benzoate

2,6

 

30

1194-65-6

2,6-Dichlorobenzonitrile

2,6

 

31

535-80-8

3-Chlorobenzoic acid

2,7

pKa = 3,82

32

108-88-3

Toluene

2,7

 

33

90-15-3

1-Naphthol

2,7

pKa = 9,34

34

608-27-5

2,3-Dichloroaniline

2,8

 

35

108-90-7

Chlorobenzene

2,8

 

36

1746-13-0

Allyl phenyl ether

2,9

 

37

108-86-1

Bromobenzene

3,0

 

38

100-41-4

Ethylbenzene

3,2

 

39

119-61-9

Benzophenone

3,2

 

40

92-69-3

4-Phenylphenol

3,2

pKa = 9,54

41

89-83-8

Thymol

3,3

 

42

106-46-7

1,4-Dichlorobenzene

3,4

 

43

122-39-4

Diphenylamine

3,4

pKa = 0,79

44

91-20-3

Naphthalene

3,6

 

45

93-99-2

Phenyl benzoate

3,6

 

46

98-82-8

Isopropylbenzene

3,7

 

47

88-06-2

2,4,6-Trichlorophenol

3,7

pKa = 6

48

92-52-4

Biphenyl

4,0

 

49

120-51-4

Benzyl benzoate

4,0

 

50

88-85-7

2,4-Dinitro-6-sec-butylphenol

4,1

 

51

120-82-1

1,2,4-Trichlorobenzene

4,2

 

52

143-07-7

Dodecanoic acid

4,2

pKa = 5,3

53

101-84-8

Diphenyl ether

4,2

 

54

85-01-8

Phenanthrene

4,5

 

55

104-51-8

n-Butylbenzene

4,6

 

56

103-29-7

Dibenzyl

4,8

 

57

3558-69-8

2,6-Diphenylpyridine

4,9

 

58

206-44-0

Fluoranthene

5,1

 

59

603-34-9

Triphenylamine

5,7

 

60

50-29-3

DDT

6,5

 

DESCRIPTION OF THE METHOD

Preliminary estimate of the partition coefficient

16. If it is necessary, the partition coefficient of the test substance may be estimated preferably by using a calculation method (see Appendix, or where appropriate, by using the ratio of the solubility of the test substance in the pure solvents.

Apparatus

17. A liquid-phase chromatograph fitted with a low-pulse pump and a suitable detection system is required. A UV detector, using a wavelength of 210 nm, or an RI detector is applicable to the wide variety of chemical groups. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have a minimal percentage of polar groups (16). Commercial microparticulate reverse-phase packing or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

Mobile phase

18. HPLC-grade methanol and distilled or de-ionised water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting substances with a log P of 6 within an hour, at a flow rate of 1 ml/min. For substances with a log P above 6 it may be necessary to shorten the elution time (and those of the reference substances) by decreasing the polarity of the mobile phase or the column length.

19. The test substance and the reference substances must be soluble in the mobile phase in sufficient concentration to allow their detection. Additives may be used with the methanol-water mixture in exceptional cases only, since they will change the properties of the column. In these cases it must be confirmed that the retention time of the test and reference substances are not influenced. If methanol-water is not appropriate, other organic solvent-water mixtures can be used, e.g. ethanol-water, acetonitrile-water or isopropyl alcohol (2-propanol)-water.

20. The pH of the eluent is critical for ionisable substances. It should be within the operating pH range of the column, usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not normally advisable since the use of an alkaline mobile phase may cause rapid deterioration in the performance of the column.

Solutes

21. The test and reference substances must be sufficiently pure in order to assign the peaks in the chromatograms to the respective substances. Substances to be used for test or calibration purposes are dissolved in the mobile phase if possible. If a solvent other than the mobile phase is used to dissolve the test and reference substances, the mobile phase should be used for the final dilution prior to injection.

Test conditions

22. The temperature during the measurement should not vary by more than ± 1 °C.

Determination of dead time to

23. The dead time t0 can be measured by using unretained organic substances (e.g. thiourea or formamide). A more precise dead time can be derived from the retention times measured or a set of approximately seven members of a homologous series (e.g. n-alkyl methyl ketones) (17). The retention times tR (nC + 1) are plotted against tR (nC), where nC is the number of carbon atoms. A straight line, tR (nC + 1) = A tR (nC) + (1 – A)t0, is obtained, where A, representing k(nC + 1)/k(nC), is constant. The dead time t0 is obtained from the intercept (1 – A)t0 and the slope A.

Regression Equation

24. The next step is to plot a correlation log k versus log P for appropriate reference substances with log P values near the value expected for the test substance. In practice, from 6 to 10 reference substances are injected simultaneously. The retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are plotted as a function of log P. The regression equation is performed at regular intervals, at least once daily, so that account can be taken of possible changes in column performance.

DETERMINATION OF THE POW OF THE TEST SUBSTANCE

25. The test substance is injected in the smallest detectable quantities. The retention time is determined in duplicate. The partition coefficient of the test substance is obtained by interpolation of the calculated capacity factor on the calibration graph. For very low and very high partition coefficients extrapolation is necessary. Especially in these cases attention must be given to the confidence limits of the regression line. If the retention time of sample is outside the range of retention times obtained for the standards, a limit value should be quoted.

DATA AND REPORTING

Test report

26. The following must be included in the report:

 if determined the preliminary estimate of the partition coefficient, the estimated values and the method used; and if a calculation method was used, its full description including identification of the data base and detailed information on the choice of fragments;

 test and reference substances: purity, structural formula and CAS number,

 description of equipment and operating conditions: analytical column, guard column,

 mobile phase, means of detection, temperature range, pH;

 elution profiles (chromatograms);

 deadtime and how it was measured;

 retention data and literature log Pow values for reference substances used in calibration;

 details on fitted regression line (log k versus log Pow) and the correlation coefficient of the line including confidence intervals;

 average retention data and interpolated log Pow value for the test substance;

 in case of a mixture: elution profile chromatogram with indicated cut-offs;

 log Pow values relative to area % of the log Pow peak;

 calculation using a regression line;

 calculated weighted average log Pow values, when appropriate.

LITERATURE

(1) C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

(2) W. Klein, W. Kördel, M. Weiss and H.J. Poremski. (1988). Updating of the OECD Test Guideline 107 Partition Coefficient n-Octanol-Water, OECD Laboratory Intercomparison Test on the HPLC Method. Chemosphere. 17, 361.

(3) C.V. Eadsforth. (1986). Application of Reverse H.P.L.C. for the Determination of Partition Coefficient. Pesticide Science. 17, 311.

(4) H. Ellgehausen, C. D'Hondt and R. Fuerer (1981). Reversed-phase chromatography as a general method for determining octan-1-ol/water partition coefficients. Pesticide. Science. 12, 219.

(5) B. McDuffie (1981). Estimation of Octanol Water Partition Coefficients for Organic Pollutants Using Reverse Phase High Pressure Liquid Chromatography. Chemosphere. 10, 73.

(6) OECD (2000). Guideline for Testing of Chemicals — Partition Coefficient (n-octanol/water): pH-metric Method for Ionisable Substances. Draft Guideline, November 2000.

(7) OSPAR (1995). ‘Harmonised Offshore Chemicals Notification Format (HOCFN) 1995’, Oslo and Paris Conventions for the Prevention of Marine Pollution Programmes and Measures Committee (PRAM), Annex 10, Oviedo, 20–24 February 1995.

(8) M. Thatcher, M. Robinson, L. R. Henriquez and C. C. Karman. (1999). An User Guide for the Evaluation of Chemicals Used and Discharged Offshore, A CIN Revised CHARM III Report 1999. Version 1.0, 3. August.

(9) E. A. Vik, S. Bakke and K. Bansal. (1998). Partitioning of Chemicals. Important Factors in Exposure Assessment of Offshore Discharges. Environmental Modelling & Software Vol. 13, pp. 529-537.

(10) L.O. Renberg, S.G. Sundstroem and K. Sundh-Nygård. (1980). Partition coefficients of organic chemicals derived from reversed-phase thin-layer chromatography. Evaluation of methods and application on phosphate esters, polychlorinated paraffins and some PCB-substitutes. Chemosphere. 9, 683.

(11) W.E. Hammers, G.J.Meurs and C.L. De-Ligny. (1982). Correlations between liquid chromatographic capacity ratio data on Lichrosorb RP-18 and partition coefficients in the octanol-water system. J. Chromatography 247, 1.

(12) J.E. Haky and A.M. Young. (1984). Evaluation of a simple HPLC correlation method for the estimation of the octanol-water partition coefficients of organic compounds. J. Liq. Chromatography. 7, 675.

(13) S. Fujisawa and E. Masuhara. (1981). Determination of Partition Coefficients of Acrylates Methacrylates and Vinyl Monomers Using High Performance Liquid Chromatography. Journal of Biomedical Materials Research. 15, 787.

(14) C. Hansch and A. J. Leo. (1979). Substituent Constants for Correlation Analysis in Chemistry and Biology. John Willey, New York.

(15) C. Hansch, chairman; A.J. Leo, dir. (1982). Log P and Parameter Database: A tool for the quantitative prediction of bioactivity — Available from Pomona College Medical Chemistry Project, Pomona College, Claremont, California 91711.

(16) R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

(17) G.E. Berendsen, P.J. Schoenmakers, L. de Galan, G. Vigh, Z. Varga-Puchony, and J. Inczédy. (1980). On determination of hold-up time in reversed-phase liquid chromatography. J. Liq. Chromato. 3, 1669.

Appendix

POW calculation methods

INTRODUCTION

1. This appendix provides a short introduction to the calculation of Pow. For further information the reader is referred to textbooks (1)(2).

2. Calculated values of Pow are used for:

 deciding which experimental method to use: Shake Flask method for log Pow between – 2 and 4 and HPLC method for log Pow between 0 and 6;

 selecting conditions to be used in HPLC (reference substances, methanol/water ratio);

 checking the plausibility of values obtained through experimental methods;

 providing an estimate when experimental methods cannot be applied.

3. The calculation methods suggested here are based on the theoretical fragmentation of the molecule into suitable substructures for which reliable log Pow increments are known. The log Pow is obtained by summing the fragment values and the correction terms for intramolecular interactions. Lists of fragment constants and correction terms are available (1)(2)(3)(4)(5)(6). Some are regularly updated (3).

4. In general, the reliability of calculation methods decreases as the complexity of the substance under study increases. In the case of simple molecules of low molecular weight and with one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured values can be expected. The margin of error will depend on the reliability of the fragment constants used, the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of correction terms. In the case of ionising substances the charge and degree of ionisation must be taken into consideration (10).

5. The hydrophobic substituent constant, π, originally introduced by Fujita et al. (7) is defined as:

πX = log Pow (PhX) – log Pow (PhH)

where PhX is an aromatic derivative and PhH the parent substance.



e.g.

πCl

= log Pow (C6H5Cl) – log Pow (C6H6)

= 2,84 – 2,13

= 0,71

The π-method is primarily of interest for aromatic substances. π-values for a large number of substituents are available (4)(5).

6. Using the Rekker method (8) the log Pow value is calculated as:

image

where ai is the number of times a given fragment occurs in the molecule and fi is the log Pow increment of the fragment. The interaction terms can be expressed as an integral multiple of one single constant Cm (so-called ‘magic constant’). The fragment constants fi and Cm have been determined from a list of 1 054 experimental Pow values of 825 substances using multiple regression analysis (6)(8). The determination of the interaction terms is carried out according to set rules (6)(8)(9).

7. Using the Hansch and Leo method (4), the log Pow value is calculated as:

image

where fi is a fragment constant, Fj a correction term (factor), ai and bj the corresponding frequency of occurence. Lists of atomic and group fragmental values and of correction terms Fj were derived by trial and error from experimental Pow values. The correction terms have been divided into several different classes (1)(4). Sofware packages have been developed to take into account all the rules and correction terms (3).

COMBINED METHOD

8. The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (3)(4) or by existing measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch- π values or with Rekker or Leo fragment constants.

Remarks:

(i) The calculation methods are only applicable to partly or fully ionised substances when the necessary correction factors are taken into account.

(ii) If the existence of intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. + 0,6 to + 1,0 log Pow units) must be added (1). Indications on the presence of such bonds can be obtained from stereo models or spectroscopic data.

(iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation.

(iv) The revisions of lists of fragment constants should be followed carefully.

LITERATURE ON CALCULATION METHODS

(1) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.). Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York (1982).

(2) W.J. Dunn, J.H. Block and R.S. Pearlman (ed.). Partition Coefficient, Determination and Estimation, Pergamon Press, Elmsford (New York) and Oxford (1986).

(3) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

(4) C. Hansch and A.J. Leo. Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York (1979).

(5) Leo, C. Hansch and D. Elkins. (1971) Partition coefficients and their uses. Chemical. Reviews. 71, 525.

(6) R. F. Rekker, H. M. de Kort. (1979). The hydrophobic fragmental constant: An extension to a 1 000 data point set. Eur. J. Med. Chem. — Chim. Ther. 14, 479.

(7) Toshio Fujita, Junkichi Iwasa & Corwin Hansch (1964). A New Substituent Constant, π, Derived from Partition Coefficients. J. Amer. Chem. Soc. 86, 5175.

(8) R.F. Rekker. The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Vol. 1, Elsevier, New York (1977).

(9) C.V. Eadsforth and P. Moser. (1983). Assessment of Reverse Phase Chromatographic Methods for Determining Partition Coefficients. Chemosphere. 12, 1459.

(10) R.A. Scherrer. ACS — Symposium Series 255, p. 225, American Chemical Society, Washington, D.C. (1984).

▼B




PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS

TABLE OF CONTENTS

GENERAL INTRODUCTION

B.1 bis.

ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE

B.1 tris.

ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD

B.2.

ACUTE INHALATION TOXICITY

B.3.

ACUTE TOXICITY (DERMAL)

B.4.

ACUTE TOXICITY: DERMAL IRRITATION/CORROSION

B.5.

ACUTE TOXICITY: EYE IRRITATION/CORROSION

B.6.

SKIN SENSITISATION

B.7.

REPEATED DOSE 28-DAY ORAL TOXICITY STUDY IN RODENTS

B.8.

SUBACUTE INHALATION TOXICITY: 28-DAY STUDY

B.9.

REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)

B.10.

MUTAGENICITY — IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST

B.11.

MUTAGENICITY — IN VIVO MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST

B.12.

MUTAGENICITY — IN VIVO MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST

B.13/14.

MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA

B.15.

MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION — SACCHAROMYCES CEREVISIAE

B.16.

MITOTIC RECOMBINATION — SACCHAROMYCES CEREVISIAE

B.17.

MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE MUTATION TEST

B.18.

DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS IN VITRO

B.19.

SISTER CHROMATID EXCHANGE ASSAY IN VITRO

B.20.

SEX-LINKED RECESSIVE LETHAL TEST IN DROSOPHILA MELANOGASTER

B.21.

IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS

B.22.