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BRITISH STANDARD Water quality Ð Gas chromatographic determination of some selected chlorophenols in water The European Standard EN 12673:1998 has the status of a British Standard ICS 13.060.50 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BS EN 12673:1999 BS 6068-2.65: 1999 Confirmed July 2008 BS EN 12673:1999 National foreword This British Standard is the English language version of EN 12673:1998 The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/2, Physical, chemical and biochemical methods, which has the responsibility to: Ð aid enquirers to understand the text; Ð present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; Ð monitor related international and European developments and promulgate them in the UK A list of organizations represented on this subcommittee can be obtained on request to its secretary BS EN 12673 is one of a series of standards on water quality, others of which have been, or will be, published as Sections of BS 6068 This standard has therefore been given the secondary identifier BS 6068-2.65 The various Sections of BS 6068 are comprised within Parts to 7, which, together with Part 0, are listed below Part Introduction Part Glossary Part Physical, chemical and biochemical methods Part Radiological methods Part Microbiological methods Part Biological methods Part Sampling Part Precision and accuracy NOTE The tests described in this British Standard should only be carried out by suitably qualified persons with an appropriate level of chemical expertise Standard chemical procedures should be followed throughout Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled ªInternational Standards Correspondence Indexº, or by using the ªFindº facility of the BSI Standards Electronic Catalogue A British Standard does not purport to include all the necessary provisions of a contract Users of British Standards are responsible for their correct application Compliance with a British Standard does not of itself confer immunity from legal obligations Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages to 16, an inside back cover and a back cover This British Standard, having been prepared under the direction of the Health and Environment Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 June 1999 BSI 06-1999 ISBN 580 30998 Amendments issued since publication Amd No Date Comments EN 12673 EUROPEAN STANDARD NORME EUROPÊENNE EUROPẰISCHE NORM December 1998 ICS 13.060.01 Descriptors: water tests, water, quality, water pollution, chemical analysis, determination of content, phenols, chromatographic analysis, gas chromatography, extraction English version Water quality Ð Gas chromatographic determination of some selected chlorophenols in water Qualite de l'eau Ð Dosage par chromatographie en phase gazeuse de certains chloropheÂnols dans les eaux Wasserbeschaffenheit Ð Gaschromatographische Bestimmung einiger ausgewaÈhlter Chlorphenole in Wasser This European Standard was approved by CEN on 26 November 1998 CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member This European Standard exists in three official versions (English, French, German) A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom CEN European Committee for Standardization Comite EuropeÂen de Normalisation EuropaÈisches Komitee fuÈr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Ref No EN 12673:1998 E Page EN 12673:1998 Foreword This European Standard has been prepared by Technical Committee CEN/TC 230, Water analysis, the Secretariat of which is held by DIN This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 1999, and conflicting national standards shall be withdrawn at the latest by June 1999 According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom Annexes designated ªinformativeº are only given for information In this standard annexes A to G are informative It is absolutely essential that tests conducted according to this standard are carried out by suitably qualified staff BSI 06-1999 Page EN 12673:1998 Scope Principle This European Standard describes the gas chromatographic determination of 19 chlorophenols (2-, 3-, and 4-chlorophenol, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dichlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6 and 3,4,5-trichlorophenol, 2,3,4,5-, 2,3,4,6-, and 2,3,5,6-tetrachlorophenol and pentachlorophenol) in drinking water, groundwater, rainwater, waste water, sea water and surface water This standard describes an acetylation followed by a liquid/liquid extraction and determination by gas chromatography and electron capture detection or mass selective detection The method is validated for drinking water, surface water and waste water, but may be used for all above mentioned types of water With this method chlorophenols can be determined over a range of concentrations from 0,1 mg/l to mg/l, depending on the quantity of sample used and the component sensitivity (level of chlorination) (see annex A) In some cases complete separation of isomers cannot be achieved Then the sum is reported This method may be applicable to other halogenated phenolic compounds, provided the method is validated for each case Chlorophenols present in the aqueous samples are derivatized with acetic anhydride to their corresponding acetates The derivatives formed are extracted from the sample with hexane The hexane fraction is analysed by gas chromatography with electron capture detection or mass selective detection Depending on the sample type pretreatment involves acid-base partition prior to the derivatization step Normative references This European Standard incorporates by dated or undated reference, provisions from other publications These normative references are cited at the appropriate places in the text and the publications are listed hereafter For dated references subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision For undated references the latest edition of the publication referred to applies ISO 5667-5:1991, Water quality Ð Sampling Ð Part 5: Guidance on sampling of drinking water and water used for food and beverage processing ISO 5667-6:1990, Water quality Ð Sampling Ð Part 6: Guidance on sampling of rivers and streams ISO 5667-8:1993, Water quality Ð Sampling Ð Part 8: Guidance on sampling of wet deposition ISO 5667-9:1992, Water quality Ð Sampling Ð Part 9: Guidance on sampling from marine waters ISO 5667-10:1992, Water quality Ð Sampling Ð Part 10: Guidance on sampling from waste waters ISO 5667-11:1993, Water quality Ð Sampling Ð Part 11: Guidance on sampling from ground waters Definitions For the purpose of this European Standard the following definition applies 3.1 chlorophenol compound having an aromatic nucleus carrying one hydroxyl group and from one to five chlorine atoms BSI 06-1999 Interferences Surfactants, emulsifiers, higher concentrations of polar solvents and other phenolic substances can affect the extractive derivatization step Suspended particles in the water can also interfere and reduce the recovery A second liquid phase in the water (e.g mineral oil compounds, highly volatile halogenated hydrocarbons, emulsified fats and waxes) disturbs sampling, sample preparation and the enrichment In those cases the examination is restricted to the aqueous phase and the portion of the non-aqueous phase is reported separately Reagents WARNING The use of this European Standard may involve hazardous materials, operations and equipment This standard does not purport to address all the safety problems associated with its use It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use 6.1 General requirements All reagents shall be of such a purity that they not give rise to significant interfering peaks in the gas chromatographic analysis of the extracts This shall be verified for each batch of material by running procedural blanks with each batch of samples analyzed Reagents shall be stored in all glass containers with glass stoppers or with polytetrafluoroethylene (PTFE) lined caps 6.2 Gas chromatographic gases, including helium, argon/methane, nitrogen or hydrogen They shall be of a purity as recommended by the gas chromatograph manufacturer 6.3 Ethanol, C2H5OH 6.4 n-Hexane, C6H14 6.5 Potassium carbonate solution, c(K2CO3) = 1,0 mol/l 6.6 Potassium carbonate solution, c(K2CO3) = 0,1 mol/l 6.7 Acetic anhydride, C4H6O3 NOTE Impurities in the acetic anhydride may affect the recovery In that case it is possible to purify acetic anhydride by distillation Page EN 12673:1998 6.8 Toluene, C7H8 6.9 Phosphoric acid, c(H3PO4) = 0,5 mol/l 6.10 Sodium sulfate, Na2SO4, anhydrous, neutral NOTE Some batches of sodium sulfate have been found to be alkaline In these circumstances it is possible to wash with methanol containing 0,5 ml concentrated hydrochloric acid per litre and to dry on a steam bath before roasting in a muffle furnace (7.6) at 500 8C ± 20 8C for h ± 0,5 h 6.11 Sodium hydroxide, c(NaOH) = 0,1 mol/l 6.12 Sodium thiosulfate pentahydrate, (Na2S2O3´5H2O), crystals or a 10 % (m/m) solution 6.13 2-chlorophenol, C6H5OCl 6.14 3-chlorophenol, C6H5OCl 6.15 4-chlorophenol, C6H5OCl 6.16 2,3-dichlorophenol, C6H4OCl2 6.17 2,4-dichlorophenol, C6H4OCl2 6.18 2,5-dichlorophenol, C6H4OCl2 6.19 2,6-dichlorophenol, C6H4OCl2 6.20 3,4-dichlorophenol, C6H4OCl2 6.21 3,5-dichlorophenol, C6H4OCl2 6.22 2,3,4-trichlorophenol, C6H3OCl3 6.23 2,3,5-trichlorophenol, C6H3OCl3 6.24 2,3,6-trichlorophenol, C6H3OCl3 6.25 2,4,5-trichlorophenol, C6H3OCl3 Prepare a mixed standard of two component solutions in such a concentration that if a small volume is added to a sample, the amount of the internal standards gives peak heights on the chromatogram in the upper part of the linear working range Typically a concentration of 10 mg/ml can be used Confirm their concentration prior to use 6.32.2 Stock solutions Prepare stock solutions of the chlorophenols by weighing each compound (6.13 to 6.31) and dissolving it in ethanol (6.3) Typical concentrations of the stock solutions are given in annex B Alternatively, commercially available standard solutions can be used Confirm the concentrations NOTE Confirmation may be accomplished by spectrometric methods (e.g UV spectrometry) or comparison with a standard of known concentration or from another source NOTE Stock solutions are stable for at least half a year when stored in the dark at 8C At a temperature of 218 8C they are stable for at least one year 6.32.3 Intermediate standards Prepare this mixed standard solution by dilution of the stock solutions (6.32.2) Suitable concentrations are given in annex B The intermediate standards should be prepared freshly every month 6.32.4 Working standards Prepare a minimum of five different concentrations by suitable dilutions of the intermediate solution (6.32.3) with ethanol (6.3) Suitable concentrations are given in annex B The working standards may be used for days 6.26 2,4,6-trichlorophenol, C6H3OCl3 Apparatus 6.27 3,4,5-trichlorophenol, C6H3OCl3 7.1 General requirements Standard laboratory glassware cleaned to eliminate all interferences 6.28 2,3,4,5-tetrachlorophenol, C6H2OCl4 6.29 2,3,4,6-tetrachlorophenol, C6H2OCl4 6.30 2,3,5,6-tetrachlorophenol, C6H2OCl4 6.31 Pentachlorophenol, C6Cl5OH 6.32 Standard solutions of chlorophenols 6.32.1 Internal standard solutions Prepare solutions of at least two internal standards in ethanol (see 6.3) For electron capture detection the following internal standard can be used: Ð 2,4-dibromophenol, C6H4OBr2; Ð 2,6-dibromophenol, C6H4OBr2; Ð 2,3,6-trichlorophenol (see 6.24), C6H3OCl3; Ð 2,4,6-tribromophenol, C6H3OBr3 For mass selective detection similar labelled compounds can be used NOTE The two internal standards are used as a control for the analytical procedure The choice of the two internal standards should reflect the anticipated occurrence of the chlorophenols in the sample (e.g if dichlorophenols are expected 2,4-dibromophenol and 2,6-dibromophenol should be used) NOTE Heating to a temperature above 150 8C before use assists in freeing glassware from possible contaminations This procedure should not be used for volumetric bottles Also an alkaline washing procedure can be used 7.2 Sample bottles, all glass, with glass stoppers or with PTFE lined caps A random bottle per batch shall be checked for interfering contamination by running a blank determination prior to use (see 9.5) 7.3 Flasks with ground stopper, glass, 100 ml 7.4 Capillary gas chromatograph, equipped with an injector system which minimizes decomposition of the sample (e.g on-column or glass-lined injector), an electron capture detector or mass selective detector and a recorder system (integrator, computer etc.) 7.5 Capillary columns, fused silica; for electron capture detection at least two with stationary phases of different polarity; for mass spectrometric detection one column suffices Typical: length 30 m, internal diameter < 0,4 mm, coated with chemically bonded methyl silicones or phenyl (5 %) methyl silicones (apolar) or cyanopropylene (14 %) methylsilicones (polar) and with a film thickness of 0,25 mm or equivalent BSI 06-1999 Page EN 12673:1998 7.6 Muffle furnace, set to 500 8C ± 20 8C 7.7 Apparatus for liquid/liquid (L/L) extraction 7.7.1 Separating funnels, 500 ml and 250 ml with grease free glass or PTFE taps 7.7.2 Shaking machine Sampling For sampling the following ISO methods are applicable: drinking water surface water rainwater sea water waste water groundwater ISO 5667-5; ISO 5667-6; ISO 5667-8; ISO 5667-9; ISO 5667-10; ISO 5667-11 The bottles shall be filled to the brim with the water sample and stoppered On sample collection, take care that no interfering substances enter the water sample, and no losses of the determinands occur This is especially important in the use of any plastic tubing used within the sampling apparatus If necessary, it shall be proved by control tests that no losses by adsorption occur Glass and stainless steel devices are preferable Some chlorophenols may degrade in an aqueous environment Therefore, unless experimental stability trials indicate otherwise, extract samples within two days of sampling If extraction is extended beyond two days this shall be noted in the test report If the interval between sampling and extraction exceeds one day, keep the samples at 8C in the dark If free halogens are suspected, add, at the time of sampling, some crystals of Na2S2O3´5H2O or 0,1 ml of a 10 % (m/m) Na2S2O3 solution (6.12) per 125 ml of sample Otherwise, not add any preservation agent Procedure 9.1 Sample pretreatment In this section two procedures are given: Ð a method including acid-base partition which may be applied for dirty samples or when enrichment of the sample is required (9.1.1); Ð a procedure employing direct acetylation suitable for relatively clean samples (9.1.2) It is permissible for sample volumes to be increased if required The volumes of all other reagents (except the internal standard) shall be adjusted accordingly Moreover, as the calibration is based on the total procedure, the volumes used for the preparation of calibration solutions shall also be adjusted accordingly Apply one of the following procedures BSI 06-1999 9.1.1 Clean up/enrichment procedure Adjust the pH of the sample to pH = by the addition of phosphoric acid (6.9) Pour 200 ml of the sample into a 500 ml separating funnel (7.7.1) Add 200 ml of internal standard (6.32.1) Extract successively with 40 ml, 40 ml and 20 ml of toluene (6.8) Shake for 10 minutes each time using the shaking machine (7.7.2) NOTE If an emulsion forms during the extraction process, the emulsion can be broken by e.g violent shaking, deep freezing, ultrasonification or separating out by means of the addition of salts Shake the collected toluene extract with a 3 20 ml 0,1 mol/l potassium carbonate solution (6.6), for minutes each time, in a 250 ml separating funnel Collect the water layers and proceed with 9.2.2 9.1.2 Pretreatment if no clean up/enrichment procedure is followed Take a sample of 50 ml or an aliquot diluted with distilled water to a volume of 50 ml Neutralize acidic samples with sodium hydroxide (6.11) to a pH value of about and alkaline samples with phosphoric acid (6.9) to a pH of about 10 Add 200 ml of internal standard (6.32.1) 9.2 Acetylation procedure 9.2.1 Acetylation of the working standards Treat each of the working standards (6.32.4) as follows Transfer with a pipette into a 100 ml open flask (7.3): Ð 50 ml of distilled water; Ð 2,00 ml of the working standard (6.32.4); Ð 200 ml of the internal standard (6.32.1) The following steps shall be carried out in the exact times given and without interruption Add ml of the mol/l potassium carbonate solution (6.5) and subsequently ml of the acetic anhydride (6.7) and stir vigorously for to allow the release of carbon dioxide NOTE This procedure can also be carried out using a separating funnel or a microseparator (see annex C) Allow to stand for 10 and then add 5,0 ml of n-hexane (6.4) Close the flask with the stopper and stir for Allow the two phases to separate Transfer as large a portion as possible of the hexane phase to a vial Dry the hexane phase with anhydrous sodium sulfate (6.10) or by freezing Store at 8C These acetylated solutions are the calibration solutions Calculate the content of each substance (mg/ml) in each of the calibration solutions NOTE The efficiency of the derivatization step may be checked with a selection of chlorophenolacetates Generally these compounds are not suitable for calibration purposes because sufficiently pure chlorophenolacetates are not always available Page EN 12673:1998 9.2.2 Acetylation of the sample Transfer the collected aqueous phases or an aliquot of 9.1.1 or the (neutralized) sample of 9.1.2 into a 100 ml open flask (7.3) and add ml of the mol/l potassium carbonate solution (6.5) Carry out the following steps in the exact times given and without interruption Determine the calibration function by regression analysis using the ratios yie/yse and rie/rse Establish the linear regression function using the pairs of ratios yie/yse and rie/ rse of the measured series in the following equation yie r = mi ie + bi (1) yse rse NOTE This procedure can also be carried out using a separating funnel or a microseparator (see annex C) Where: Add ml of acetic anhydride (6.7) Stir vigorously for to allow the release of carbon dioxide Allow to stand at room temperature for 10 and add 5,0 ml of n-hexane (6.4) Close the flask with the stopper and stir for Allow the phases to separate Remove the water layer and dry the hexane phase with anhydrous sodium sulfate (6.10) or by freezing NOTE If an emulsion forms during the extraction process, the emulsion can be broken by e.g violent shaking, deep freezing, ultrasonification or separating out by means of the addition of salts In case of emulsification recoveries should be checked yie yse rie rse 9.3 Calibration 9.3.1 Gas chromatograph calibration Set up the gas chromatographic instrument, equipped with the columns (7.5), according to the manufacturer's instructions Optimize gas flows Ensure it is in a stable condition Guidance on the initial gas chromatographic conditions is given in annex D Calibrate by direct injection of the acetylated working standards (9.2.1) and in addition run a blank Measure the gas chromatographic signals for each substance against concentration This gives information on retention times and relative responses of the determinands and the linear working range of the gas chromatograph and detector NOTE Chromatograms of standards should be checked for retention time and peak resolution changes, and losses caused by decomposition within the injection liner NOTE Separation can be considered as satisfactory if the height measured from the base line of the trough between the two adjacent peaks is no more than 20 % of the height of the highest peak; the peaks in this instance need to be of comparable height Separation between 2,3,4,5-tetrachlorophenol acetate and 2,3,4,6-tetrachlorophenol acetate can be critical The resolution should at least be 0,5 Generally the acetates of 2,4and 2,5-dichlorophenol are not separated 9.3.2 Calibration of the procedure For explanation of the subscripts used see Table Table Ð Explanation of the subscripts Subscript Meaning i Identity of the substance e Calibration s Internal standard mi bi is the measured value of the determinand i as e.g peak height or peak area; is the measured value of the internal standard s as e.g peak height or peak area; is the mass concentration of the determinand i in the calibration solution in micrograms per litre; is the mass concentration of the internal standard s in the calibration solution in micrograms per litre; is the slope of the calibration function, also called the response factor; is the intercept of the calibration function with the ordinate as e.g peak height or peak area 9.4 Measurement Prepare gas chromatograms of the extracts obtained in 9.2.2 by injecting a defined volume, typically ml to ml (but the same volume as in 9.3.1), into the gas chromatograph This procedure shall be performed by analysing the samples on the two capillary columns of a differing polarity (7.5) The following measurement conditions shall be observed in the detection of substances using a mass spectrometer Ionization procedure: electron ionization, electron energy at least 45 eV Mass range during registration of the spectra: 46 to 280 absolute mass units (u), at least 10 u above the highest molecular mass of the substances in question If there is interference e.g due to CO2, the spectra registration can be begun at 46 u Cycle time: < sec ± at least spectra should be registered for each substance peak If, with increased sensitivity only selected ions are detected register the base peak with additional ions (as they appear in the spectra) with the same cycle time as above BSI 06-1999 Page EN 12673:1998 9.5 Quality control experiment For the quality control of the analytical procedure take the following steps Determine the substance specific blanks by running the background gas chromatograms of the respective total method as applied to a sample of interference free water (i.e pretreatment, extraction, purification, gas chromatography) If blank values are unusually high (more than 10 % of the lowest measured values) every step in the procedure shall be checked in order to find the reason for these high blanks If samples concentrations are close to the limit of determination, however, blank values higher than 10 % of the lowest measured value have to be tolerated When the blank value significantly differs from the intercept of the calibration curve the cause shall be determined The minimum validity of the calibration shall be checked with every batch of samples Inject two standard extracts, one at approx 20 % and the other at approx 80 % of the selected linear working range Repeat the injections once Compare the means of the two concentrations with the calibration curve If the values are within the confidence interval of the corresponding values used in the procedure, it is permissible to use them as a calibration curve If not, check the entire procedure and establish a complete new calibration curve 10 Expression of results 10.1 Interpretation and quantification 10.1.1 GC-ECD The following steps shall be done for each column separately By means of the absolute retention times, identify the peaks of the internal standards For the remaining relevant peaks of the gas chromatograms, determine the relative retention time as compared with both internal standards Consider that a compound has been shown if the relative retention time differs by less than 0,2 % from the relative retention time obtained as in 9.3.1 The chlorophenols are quantified by using an internal standard added to the sample Errors can occur when an interfering compound co-elutes with the internal standard in the chromatogram of the extract For this reason at least two internal standards are used to determine whether interfering compounds are present or absent This presence or absence of interfering compounds can be determined from the measured responses of the internal standards When no interfering compounds are present in the extract, the ratio between the responses of the internal standard is equal to that of the ratio in the working standard The quotient of these ratios is called the relative response ratio, RRR BSI 06-1999 When no interfering compounds are present in the extract the value of RRR is in principle 1,00 In this standard it is assumed that no interfering compounds are present in the extract when RRR = 1,00 ± 0,10 When the value of RRR deviates from 1,00 ± 0,10 the response of one of the internal standards is influenced by an interfering compound present in the extract In that case the chlorophenols are quantified by using the undisturbed internal standard 10.1.2 GC-MS Identify the peaks by means of retention times as described in 10.1.1 Information on characteristic ions is given in annex F When the full scan mode is used correct the spectra by background substraction Identify the compounds by matching the spectra from the sample with the spectra of the reference substances taking into account the limits given in the following clauses Produce all spectra under the same instrumental conditions The individual reference spectra shall be created by each individual laboratory on the same GC-MS system used for the samples The reference spectra may be stored in a spectra library or derived from the corresponding calibration In the case of acquiring selected ions (SIM mode), at least three characteristic ions shall be used (See annex F.) The signal-to-noise ratio of the least intensive ion should be at least (S/N > 3) The ratio of the three masses in a spectra shall be evaluated from the mass peak height scanned at the peak maximum applying identical measurement conditions with sample and reference substance The ratio of abundance of the two less intensive ions to the base peak shall not deviate by more than 10 % between these acquisitions Structural isomers producing similar mass spectra can only be identified clearly if their GC retention times are sufficiently different Acceptable resolution is achieved if the height of the valley between two peaks is less than 25 % of the average height of the two peaks Otherwise, structural isomers are identified as isomeric pairs In general, all ions present above 10 % relative abundance in the mass spectra of the standard should be present in the mass spectra of the sample component The abundance between different ions (intensity ratio) shall agree within 20 % (absolute) between the sample and reference spectra At least three most important ions (see annex F) should be used for this test Page EN 12673:1998 10.1.3 Calculation Calculate the mass concentration of the substance using equation (2) [following the solution of equation (1)] yi bi ys rs ri = (2) f mi ys rs ri mi bi f Table Ð Rounding of results concentration (mg/l) greater than 0,01 0,1 Where: yi Round off the results as in Table is the measured value of the determinand i as e.g peak height or peak area; is the measured value of the internal standard in the sample as e.g peak height or peak area; is the mass concentration of the internal standard in the sample e.g in micrograms per litre; is the mass concentration of substance i, e.g in micrograms per litre; is the slope of the calibration function; is the intercept of the calibration function with the ordinate as e.g peak height or peak area; concentration factor; for the procedure with clean-up/enrichment (9.1.1); for direct procedure (9.1.2) Using mass spectrometry take for yi or ys respectively the peak height or peak area of the most intensive (fragment-) mass (base peak) from the corresponding substance's spectrum 10.2 Results When using electron capture detection employing two gas chromatographic methods the application of the calculation method (10.1) provides one individual result for each column used Derive the final quantitative result from these results as follows: Ð take the arithmetic mean, provided that the differences between the individual results are less than 10 % of the lowest result; Ð choose the smallest value in the event of larger differences The larger values can be the result of peak overlap Such results shall be labelled as measured values obtained from a single separation only For both the MS result obtained from a single column and the final quantitative ECD result report the mass concentrations of the substances to not more than two significant figures concentration (mg/l) up to and including 0,1 10 round up to (mg/l) 0,001 0,01 0,1 10.3 Precision In November 1996 an interlaboratory trial was carried out in which 24 laboratories from different countries took part The comparison was conducted on three types of water; drinking water, surface water and waste water In Tables 3, and information on the reproducibility and repeatability on the three water types is given Information on the lowest detected concentration is given in annex A 11 Test report The following information shall be included in the report: a) a reference to the present European Standard; b) the data required for identification of the sample examined; c) the interval between sampling and extraction; d) if stabilization by sodium thiosulfate is applied; e) the types of columns and gas chromatography conditions employed; f) the concentration of each of the chlorophenols, in micrograms per litre; g) any special circumstances observed during the determination, such as for instance other peaks observed in the chromatogram; h) all operations (e.g the settling or filtration of the sample) not prescribed in the standard which might have affected the result; i) detection with ECD or MS; For detection with MS: Identification through the registration of complete mass-spectra (SCAN mode) or individual registration of selected masses (SIM mode); number of registered or examined ion masses (given in annex F); possible occurrence of divergence of the experimental expected isotope/fragment ion-ratio; quantification mass BSI 06-1999 Page EN 12673:1998 Table Ð Performance data for drinking water (high and low concentration) Sample type Phenol Drinking water Low Drinking water High n p o x p Sr % Sr mg/l SL % SL mg/l SR % SR mg/l is is is is is is is is is is is BSI 06-1999 the the the the the the the the the the the 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro n p 51 15 34 11 55 16 50 o x p % mg/l mg/l Sr SL % SR % mg/l mg/l % mg/l 14,89 16,50 15 2,185 33 4,866 36 5,295 5,77 3,18 41 2,354 121 7,009 128 7,398 5,9 1,60 1,60 14 0,231 47 0,747 49 0,782 14 6,7 1,30 1,14 20 0,257 43 0,564 48 0,624 46 54 12 15 7,7 0,80 1,24 0,89 19 1,28 21 0,154 0,264 21 0,169 45 0,556 27 0,22 50 0,62 27 43 37 13 11 11,1 13,3 0,15 0,21 0,23 0,15 15 0,18 12 0,20 20 0,022 0,026 0,047 49 0,074 36 0,077 23 0,054 57 0,086 41 0,088 31 0,072 25 44 55 12 16 22,2 0,08 0,10 0,11 0,13 20 22,76 27,50 22 0,008 0,022 5,045 45 0,037 24 0,028 33 7,584 47 0,039 45 0,051 41 9,229 33 10 16,7 10,10 10,61 0,788 38 3,888 40 4,034 56 16 5,9 3,27 3,21 0,27 44 1,453 45 1,478 55 54 16 16 15 16,7 5,41 5,11 0,08 6,50 10 6,24 18 0,08 0,516 0,913 0,008 32 1,722 34 1,744 39 0,032 36 1,974 38 1,966 40 0,033 49 47 58 14 14 17 6,7 6,7 4,68 1,63 0,87 5,11 1,94 10 0,90 17 0,229 0,158 0,145 21 0,996 27 0,432 26 0,229 22 1,048 29 0,467 32 0,277 47 46 58 14 14 16 17,6 0,90 2,47 0,98 0,93 2,91 1,26 0,059 0,198 0,082 44 0,395 30 0,747 28 0,272 45 0,406 33 0,818 35 0,345 5,9 number of values number of data sets, i.e number of laboratories percentage of outliers general mean, without outliers indicator value relative repeatability standard deviation repeatability standard deviation relative between-laboratory standard deviation between-laboratory standard deviation relative reproducibility standard deviation reproducibility standard deviation 8 Page 10 EN 12673:1998 Table Ð Performance data for surface water (high and low concentration) Sample type Surface water High Surface water Low Phenol 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro n p 50 48 14 14 56 58 16 16 50 o x p % mg/l mg/l 12,5 12,5 Sr % 91,31 110,02 12 78,82 97,64 SL mg/l SR % mg/l % mg/l 10,67 6,065 29 28 26,13 21,82 30 29 27,13 22,73 5,9 21,51 11,06 28,06 10,69 13 1,969 1,468 33 43 7,18 4,763 14 12,5 44,57 57,17 3,362 36 50 14 6,7 8,04 8,90 0,544 21 1,689 24 1,914 15 52 56 15 16 12,5 6,3 5,9 0,10 14,99 8,83 0,09 19,45 9,04 9E-04 1,027 0,437 74 34 26 0,074 5,083 2,29 74 35 27 0,074 5,181 2,364 51 44 60 15 13 17 11,8 7,1 2,81 8,29 5,44 2,78 15 9,71 6,31 0,416 0,419 0,468 39 27 29 1,092 2,25 1,586 42 28 31 1,168 2,338 1,7 50 14 12,5 25,16 33,01 11 2,712 32 7,939 33 8,401 32 10 9,1 4,40 6,37 13 0,561 40 1,744 42 1,852 56 16 5,9 3,29 3,21 0,239 45 1,494 46 1,522 58 16 2,10 2,29 13 0,269 36 0,765 44 0,917 48 53 13 15 13,3 1,51 2,29 1,78 10 2,55 14 0,156 0,313 21 45 0,318 1,021 25 47 0,379 1,069 27 57 44 16 12 10,0 14,3 0,32 0,39 0,40 0,29 0,36 12 0,40 12 0,027 0,047 0,049 65 25 14 0,207 0,099 0,055 65 29 24 0,209 0,113 0,096 32 46 13 10,0 7,1 0,25 0,20 0,19 0,25 17 0,023 0,034 63 21 0,16 0,042 89 37 0,226 0,073 16,06 34 45 37 7,392 4,941 16,59 For the explanation of symbols see Table BSI 06-1999 Page 11 EN 12673:1998 Table Ð Performance data for waste water (high and low concentration) Sample type Waste water High Waste water Low Phenol 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro 2342, 32, 42, 52, 63, 43, 52, 3, 42, 3, 52, 3, 62, 4, 52, 4, 63, 4, 52, 3, 4, 52, 3, 4, 62, 3, 5, 6pentachloro n o x p % mg/l mg/l 48 52 13 15 56 50 16 14 52 14 50 14 6,7 7,77 8,90 14 46 56 13 16 16,7 13,3 0,09 14,15 9,19 0,09 19,45 9,04 47 41 50 14 12 14 12,5 7,7 12,5 46 13 19 Sr SL SR % mg/l % mg/l % mg/l 5 4,568 3,437 34 32 29,84 19,89 35 33 30,75 20,81 13,3 6,3 87,58 110,02 62,62 97,64 12,5 21,78 11,01 28,06 10 10,69 2,194 0,944 35 49 45,48 57,17 11 5,087 40 0,674 20 1,553 22 1,706 0,001 0,65 0,776 54 41 31 0,047 5,746 2,825 54 41 32 0,047 5,852 2,947 2,69 8,18 4,98 2,78 36 9,71 6,31 0,97 0,504 0,465 42 35 24 1,121 2,893 1,172 56 36 26 1,492 2,936 1,307 7,1 18,47 27,50 10 1,789 30 5,549 36 6,604 12,5 6,06 10,61 0,24 81 4,931 82 4,943 52 15 6,3 3,19 3,21 12 0,37 53 1,685 54 1,723 54 50 23 15 14 6,7 22,2 4,37 4,61 0,11 6,50 17 6,24 14 0,08 0,725 0,65 0,007 35 33 47 1,531 1,537 0,05 39 37 48 1,709 1,713 0,051 50 52 56 14 15 16 6,7 4,48 1,60 0,95 5,11 1,94 13 0,90 20 0,297 0,212 0,191 29 35 37 1,32 0,564 0,35 30 38 40 1,353 0,606 0,383 47 46 54 14 13 15 17,6 11,8 0,83 2,43 0,92 0,93 12 2,91 25 1,26 19 0,097 0,599 0,171 34 28 15 0,282 0,68 0,14 36 38 34 0,3 0,918 0,311 For the explanation of symbols see Table BSI 06-1999 p 5,9 7,723 5,423 18,37 37 50 43 8,018 5,525 19,44 Page 12 EN 12673:1998 Annex A (informative) Figure A.1 Ð Indication of the lowest detected concentration Lowest concentrations detected in real samples by the participants in the interlaboratory trial These data are only given as guidance The actual LOD achievable will have to be established in each particular laboratory Annex B (informative) Table B.1 Ð Typical concentrations of the standard solutions Stock solution (6.32.2) Intermediate standards (6.32.3) 30 mg/ml Working standards (6.32.4) monochlorophenol mg/ml 0,03 mg/ml to mg/ml dichlorophenols 400 mg/ml mg/ml 0,004 mg/ml to 0,5 mg/ml trichlorophenols 300 mg/ml mg/ml 0,003 mg/ml to 0,5 mg/ml tetrachlorophenols 200 mg/ml mg/ml 0,002 mg/ml to 0,5 mg/ml pentachlorophenol 100 mg/ml mg/ml 0,001 mg/ml to 0,5 mg/ml NOTE The dilution factor for the preparation of the intermediate standards of the monochlorophenols is 3:100 For the other chlorophenols this factor is 1:100 These values are given as an indication, the actual choice should reflect the anticipated occurrence of the analytes in the sample, as well as the linear range of the detector used BSI 06-1999 Page 13 EN 12673:1998 Annex C (informative) Dimensions in millimetres All dimensions are approximate Figure C.1 Ð Microseparator BSI 06-1999 Page 14 EN 12673:1998 Annex D (informative) Guideline for gas chromatography conditions The chromatographic separation can be optimized starting from the following guidelines using the calibration solutions (9.2.1) Injection temperature1: 250 8C Oven temperature: 50 8C during 50 8C to 112 8C at 8C/min 112 8C during 112 8C to 170 8C at 8C/min 170 8C during 170 8C to 280 8C at 20 8C/min 280 8C during columns: A 14% CNRPH Me Siloxane (HP-1701) B Crosslinked 5% PhMe Silicone (HP-5) both fused silica, 30 meter, 0,32 mm I.D film thickness 0,25 mm Detector: ECD Detector temperature: 300 8C Carrier gas: helium Gas flow: 20 cm/s to 30 cm/s Annex E (informative) External calibration procedure In the interlaboratory trial the calibration using both the internal standard procedure and the external standard procedure were tested It proved that the external standard procedure gave deviating results E.1 Treatment of the working standards Treat the working standards according to 9.2.1 omitting the addition of the internal standard solution E.2 Treatment of the samples Treat the samples according to either 9.1.1 or 9.1.2 depending on whether or not clean-up or enrichment is required, omitting the addition of the internal standard solution Continue with 9.2 E.3 Calibration of the procedure using external standards Establish the linear regression function using the pairs of values yie and rie of the measured series in the following equation yie = mi rie + bi (E.1) Where: yie rie mi bi E.4 Interpretation Compare the gas chromatogram obtained for the sample extract to the ones obtained with the acetylated standards If no peaks appear at the specific retention times in the chromatogram of the sample extract consider the compound as not detected When electron-capture detection is employed compare the retention times in both chromatograms When peaks appear in both chromatograms at the specific retention times consider the presence of the compounds as highly probable If mass spectrometric detection is employed proceed according to 10.1.2 E.5 Calculation Calculate the mass concentration of the substance using equation (E.2) [following the solution of equation (E.1)] y bi ri = i (E.2) f mi Where: yi ri mi bi f is the measured value of the determinand i as e.g peak height or peak area; is the mass concentration of the determinand i in the calibration solution in micrograms per litre; is the slope of the calibration function, also called the response factor; is the intercept of the calibration function with the ordinate as e.g peak height or peak area is the measured value of the determinand i as e.g peak height or peak area; is the mass concentration of substance i, e.g in micrograms per litre; is the slope of the calibration function; is the intercept of the calibration function with the ordinate as e.g peak height or peak area concentration factor; for the procedure with clean-up/enrichment (9.1.1); for direct procedure (9.1.2) Applicable only with split/splitless injection BSI 06-1999 BSI 06-1999 Annex F (informative) Table F.1 Ð Characteristic ions for MS detection Component Formula Molecular mass Isotopes (M+n)+ Molecular ion M+ relative intensity n=2 relative intensity n=4 relative intensity Fragmentation fragment M242 relative intensity fragment (M+2)242 relative intensity fragment (M+4)242 relative intensity 2-Chlorophenolacetate C8H7O2Cl 170,54 170 10 172 128 100 130 34 3-Chlorophenolacetate C8H7O2Cl 170,54 170 14 172 4,5 128 100 130 29 4-Chlorophenolacetate C8H7O2Cl 170,54 170 13 172 128 100 130 35 2,6-Dichlorophenolacetate C8H6O2Cl2 205,04 204 4,9 206 3,3 208 0,6 162 100 164 67 166 11 2,4-Dichlorophenolacetate C8H6O2Cl2 205,04 204 206 208 0,8 162 100 164 66 166 11 2,5-Dichlorophenolacetate C8H6O2Cl2 205,04 204 3,5-Dichlorophenolacetate C8H6O2Cl2 205,04 204 28 206 2,3-Dichlorophenolacetate C8H6O2Cl2 205,04 204 13 206 3,4-Dichlorophenolacetate C8H6O2Cl2 205,04 204 2,4,6-Trichlorophenolacetate C8H5O2Cl3 239,48 238 2,3,6-Trichlorophenolacetate C8H5O2Cl3 239,48 238 2,3,5-Trichlorophenolacetate C8H5O2Cl3 239,48 238 8,9 240 2,4,5-Trichlorophenolacetate C8H5O2Cl3 239,48 238 9,2 2,3,4-Trichlorophenolacetate C8H5O2Cl3 239,48 238 3,4,5-Trichlorophenolacetate C8H5O2Cl3 239,48 206 19 9,2 162 164 relative intensity 166 208 0,6 162 100 164 66 166 11 208 1,9 162 100 164 66 166 11 206 11 208 1,9 162 100 164 66 166 11 240 242 2,2 196 100 198 98 200 30 202 240 16 242 5,5 196 100 198 100 200 31 202 3,5 9,9 242 2,3 196 100 198 100 200 31 202 3,5 240 9,9 242 2,3 196 96,1 198 100 200 30 202 3,3 240 7,7 242 1,9 196 100 198 98 200 31 202 3,5 238 11,4 240 11,9 242 4,9 196 100 198 100 200 31 202 2,3,4,5-Tetrachlorophenolacetate C8H4O2Cl4 273,93 272 274 276 2,3,5,6-Tetrachlorophenolacetate C8H4O2Cl4 273,93 272 5,9 274 7,9 276 4,3 230 79 232 100 234 46 236 10 2,3,4,6-Tetrachlorophenolacetate C8H4O2Cl4 273,93 272 1,6 274 3,1 276 1,5 230 78 232 100 234 47 236 10 Pentachlorophenolacetate 306 1,6 308 2,8 310 2.3 264 66 266 100 268 65 270 22 C8H3O2Cl5 308,37 17 208 fragment (M+6)242 5,6 15 230 232 234 3,5 236 Page 15 EN 12673:1998 Page 16 EN 12673:1998 Annex G (informative) Bibliography [1] Wegman R.C.C., Hofstee A.W.M., Water Research, Vol 13, p 651-657 ªChlorophenols in surface waters of the Netherlands (1976-1977)º [2] Abrahamsson K., Xie T.M., J Chromatogr., 279 (1983), p 199 [3] Starck B., Bethge P-O., Gergov M and Talka E., Paperi ja Puu ± Papper och TraÈ, 12 (1985), p 745 EN 25667-1:1994, Water quality Ð Sampling Ð Part 1: Guidance on the design of sampling programmes (ISO 5667-1:1980) EN 25667-2:1993, Water quality Ð Sampling Ð Part 2: Guidance on sampling techniques (ISO 5667-2:1991) ENV ISO 13530, Water quality Ð Guide to analytical quality control for water analysis (ISO/TR 13530:1997) BSI 06-1999 blank BS EN 12673:1999 BS 6068-2.65: 1999 BSI 389 Chiswick High Road London W4 4AL | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BSI Ð British Standards Institution BSI is the independent national 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