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1、INTERNATIONAL STANDARD IS0 6468 First edition 1996-12-15 Water quality - Determination of certain organochlorine insecticides, polychlorinated biphenyls and chlorobenzenes - Gas chromatographic method after liquid-liquid extraction Qua/it6 de I eau - Dosage de certains insecticides organochlorks, de
2、s polychlorobiphknyles et des chlorobenz below concen- trations of 10 rig/l, special care is necessary. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editi
3、ons indicated were valid. All standards are subject to revision, and parties to agreements based on the International Standard are encouraged to in- vestigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and IS0 maintain registers of currently
4、 valid Inter- national Standards. IS0 5667-i :1980, Water quality- Sampling - Part 1: Guidance on the design on sampling programmes. IS0 5667-2:1991, Water quality - Sampling - Part 2: Guidance on sampling techniques. 3 Principle Liquid-liquid extraction of organochlorine insecticides, chlorobenzene
5、s and PCBs by an extraction solvent. After the concentration of the components with low volatility and after any clean-up steps which may be necessary, the sample extracts are analysed by gas chromatography, using an electron-capture detector. 1 Copyright International Organization for Standardizati
6、on Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/18/2007 19:40:39 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 6466: 1996(E) Table 1 - Detection limits Acronyms Chemical names (IUPAC) Detection limits Organoc
7、hlorine insecticides: HCH 1, 2, 3, 4, 5. 6-hexachlorocyclohexane, five stereoisomers: alpha-HCH beta-HCH Lindane gamma-HCH defta-HCH epsilon-HCH o,p -DDE 1 ,l -dichloro-2-(2-chlorophenyl l )-2-(4-chlorophenyl)ethylene n,p -DDE 1 ,l-dichloro-2,2-bis(4-chlorophenyl)ethylene 1 rig/l ,p -TDE 1 ,l-dichlo
8、ro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (= o,p -DDD) to up -TDE 1 ,l -dichloro-2, 2-bis(4-chlorophenyl)ethane (= p,p -DDD) 10 rig/l Y,P -DDT 1 ,l ,l-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyI)ethane depending YQ -DDT 1 ,l ,l -trichloro-2,2-bis(4-chlorophenyl)ethane on the Methoxychlor 1 ,
9、l ,l -trichloro-2,2-bis(4-methoxyphenyl)ethane compound Ndrin (1 R, 4S, 4a.X 5S, 8R, 8aR)-1, 2, 3, 4, 10, lo-hexachloro-l , 4, 4a, 5, 8, 8a-hexahydro-l , 4: 5.8-dimethanonaphthalene Dleldrln (lR, 4S, 4aS, 5R, 8R, 7S, 8S, 8aR)-1,2,3,4,10,10-hexachloro-1,4,4a, 5, 6,7,8,8a-octahydrod, 7-epoxy-1,4: 5,8-
10、dimethanonaphthalene Endrtn (lR, 4S, 4aS, 5S, 6S, 7R, 8R, 8aR)-1,2,3,4.10,10-hexachloro-1,4,4a, 5, 6,7,8,8a-octahydro-6,7-epoxy-1,4: 5,8-dimethanonaphthalene Heptachlorl) 1,4,5.6,7.8,8-heptachloro-3a, 4,7,7a-tetrahydro-4,7-methanoindenel) Heptachlor-epoxide 1, 4, 5, 6, 7, 8, 8-heptachloro-2.3-epoxy-
11、3a,4,7,7a-tetrahydro-4,7 -methanoindane Endosulfanl) 2) lf4,5,6,7,7,7-hexachloro-8,9.l0-trinorborn-5-en-2, 3-ylene-dimethyl- enesulfite: alpha-Endosulfan beta-Endosulfan Chlorobenzenes: TrCB trichlorobenzene 1 n TeCB tetrachlorobenzene to PeCB pentachlorobenzene lOngA HCB hexachlorobenzene depending
12、 on PCNB (Quintozene) pentachloronitrobenzene the compound Polychlorlnated biphenyls: PCB 28 2,4,4 -trichlorobiphenyl PCB 52 2,2 , 5,5 -tetrachlorobiphenyl 1 n PCB 101 2,2 , 4,5,5 -pentachlorobiphenyl to PCB 138 2,2 , 3,4,4 , 5 -hexachlorobiphenyl 50 rig/l PCB 153 2,2 , 4,4 , 5, S-hexachlorobiphenyl
13、 depending on PCB 180 2,2 , 3,4,4 , 5,5 -heptachlorobiphenyl the compound PCB 194 2,2 , 3,3 , 4,4 , 5, S-octachlorobiphenyl 1) The analysis of a and /3 - endosulfan as well as heptachlor requires special care due to its low stability. 2) The name endosulfan” is not acceptable for use in Italy, as it
14、 is in conflict with a trade mark registered there. 2 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/18/2007 19:40:39 MDTNo reproduction or networking permitted without license from IHS
15、 -,-,- (23 IS0 IS0 6468: 1996(E) Any substance capable of producing a response on the electron-capture detector, at a retention time indis- tinguishable from any compound of interest, will interfere. In practice, many potentially interfering sub- stances will be removed during the extraction and cle
16、an-up procedures. NOTE 1 In general, the use of two capillary columns of different polarity is sufficient for the organochlorine com- pounds analysed according to this International Standard. The results so calculated should be considered as the maximum concentrations, possibly still influenced by c
17、oelut- ing substances. It is possible that there will be cases where a more definite identification is required. 4 Reagents and materials All reagents shall be sufficiently pure to not give rise to significant interfering peaks in the gas chromatograms of the blanks. The purity of reagents used in t
18、he pro- cedure shall be checked by blank determinations (7.6). NOTE 2 Commercial “pesticide grade” solvents are avail- able. The use of these products is recommended only after verifying their quality. The quality of a solvent is checked by evaporation of about 200 ml down to 1 ml and analysis of th
19、e concentrate to determine the compounds subsequently analysed. The solvent should be considered acceptable if it does not give any detectable interfering peaks in the chro- matogram for the substance of interest. 4.1 Water purified, for example, using ion-exchange or carbon-column adsorption. 4.2 E
20、xtraction solvent. Hexane, petroleum ether or heptane are suitable. NOTE 3 Any other solvents meeting the requirements of 8.3 (recovery rate 2 60 %) may be used. muffle furnace. Cool to about 200 “C in the furnace and then to ambient temperature in a desiccator. Store in a sealed glass container. 4.
21、6 Deactivated alumina. Weigh a portion of dry alumina (4.5) into a sealable all- glass container and add 7 % C 0,2 % (m/m) of water (4.1). Seal and agitate for at least 2 h to ensure uni- formity. Store in a sealed glass container. Once the seal has been broken, storage time is nor- mally about one
22、week. After the maximum storage time, reprocess batches as described in 4.5 and this subclause. 4.7 Alumina/silver nitrate. Dissolve 0,75 g k 0,Ol g of silver nitrate in 0,75 ml f 0,Ol ml of water (4.1) using a microburette. Add 4,0 ml + 0,2 ml of acetone followed by 10 g * 0,2 g of deactivated alum
23、ina (4.6). Mix thoroughly by shaking in an open-topped conical flask, protected from light. Allow the acetone to evaporate at room temperature and prevent condensation, for example by warming with the hand. Store in the dark and use within 4 h after preparation. 4.8 Silica gel, of particle size 63 p
24、m to 200 pm, heated at 500 “C f 30 “C in batches not larger than 500 g, for about 14 h. Cool to about 200 “C in the fur- nace and then to ambient temperature in a sealed flask which is placed in a desiccator without desiccant. Use this material within one week. Deactivate the sil- ica gel by weighin
25、g a suitable quantity of silica and adding 3 % (r - extraction in the sample container with a magnetic stirrer or a high-speed stirrer and separation by a microseparator (7.2.2). NOTE 7 Depending on the method used, varying recov- eries and reproducibilities may be obtained. The yields of the select
26、ed method should be checked by the laboratory (8.3). It is recommended to perform the extraction in the sample container. Usually, a sample volume of about 1 litre is used. 7.2.1 Extraction by shaking the sampling bottle and separation in a separating funnel Add 30 ml of the extraction solvent (4.2)
27、 to the sample (7.1) and shake for at least 10 min. Transfer to a separating funnel of suitable capacity (5.3) and allow the phases to separate. Run the lower aqueous phase back into the sample container. Repeat the extraction twice with 20 ml to 30 ml of the extraction solvent (4.2). Dry the extrac
28、t using one of the following procedures: - Pass the extract through a drying column (5.9) containing anhydrous sodium sulfate (4.3), pre- viously washed with the solvent (4.2) and collect the eluate in the evaporating vessel. NOTE 8 It is advisable to wash the column with a further 10 ml to 20 ml po
29、rtion of the solvent (4.2) to obtain a better recovery. Collect the washings in the evaporating vessel. 7 Procedure 7.1 Sample pretreatment Sample pretreatment is not normally necessary. If the sample container is filled up to the ground-glass joint, shake and pour off 30 ml to 100 ml of the sample
30、in order to obtain sufficient free volume for the sub- sequent addition of the solvent. Or - Add anhydrous sodium sulfate (4.3) to the flask. Shake for 1 min. Leave for 5 min and decant the extract into the concentration apparatus. The so- dium sulfate is washed with a further 10 ml to 20 ml of solv
31、ent (4.2) and the washings added to the evaporating vessel. Or 5 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/18/2007 19:40:39 MDTNo reproduction or networking permitted without licen
32、se from IHS -,-,- IS0 6468:1996(E) IS0 - Freeze the extract at - 18 “C for 2 h. The solvent extract is decanted from the ice and transferred to the evaporating vessel. The ice is washed with a further 10 ml of solvent (4.2) and the washings are added to the evaporating vessel. 7.2.2 Extraction with
33、a magnetic or a high-speed stirrer and separation in a microseparator Add 20 ml to 30 ml of the extraction solvent (4.2) to the sample (7.1). With a magnetic stirrer and a stirring bar (5.4), stir for at least 10 min, at a speed of at least 1 000 r/min (the solvent needs to be dispersed finely in th
34、e water) keeping the sample covered, and then allow the phases to separate. Alternatively, if a high-speed stir- rer (5.4) is used, stir for 2 min while keeping the sam- ple covered at a temperature of 4 “C and allow the phases to separate. Assemble the microseparator (5.5); pour purified water (4.1
35、) into the funnel until the surface of the organic phase rises sufficiently for the extract to be withdrawn with a pipette. capture detector. 0,l ml of a solution containing 20 g/l of decane or dodecane in hexane are added to the extract to be concentrated. 7.3.2 Concentration using a rotary evapora
36、tor Concentrate the extract in a tapered flask, or prefer- ably, in a tapered flask with an ampoule extension on a rotary evaporator (5.8) to not less than 0,6 ml at a constant vacuum of greater than 340 mbar. A Kuderna-Danish evaporation flask (5.6) is mounted between the evaporating vessel and the
37、 rotary evapo- rator. Place the evaporating vessel with the solvent extract in an unheated water bath or, for higher boiling extrac- tants, in a water bath at a temperature not exceeding 50 “C. When the concentration is finished, quantitat- ively transfer the extract into a 1 ml measuring flask. Car
38、efully rinse the walls of the evaporating vessel with a small volume of solvent (4.2). Transfer the rinsings to the measuring flask and fill up to volume with the solvent. Dry the extract as described in 7.2.1. 7.4 Gas chromatography 7.3 Concentration of the extract Concentrate the combined dried ex
39、tracts from either 7.2.1 or 7.2.2 by either of the procedures described in 7.3.1 or 7.3.2 or by any other suitable system (5.8). Ensure that no significant losses of the more volatile determinands of interest occur. 7.3.1 Concentration using a Kuderna-Danish evaporator Good detection limits can be o
40、btained by evaporating the sample extract to a small volume with the Kuderna-Danish evaporator (5.6) and a Snyder micro- column (5.7) as follows. Collect the dried extract in a Kuderna-Danish evapor- ator. Add two anti-bumping granules (4.11) and evaporate to 5 ml f 1 ml on a steam bath. Further con
41、centrate the extract to less than 1 ml using a Snyder microcol- umn or a gentle stream of clean inert gas (e.g. nitro- gen) with a tube placed in a warm water bath (not exceeding 40 “C). NOTE 9 No further precautions are necessary if the ex- tract is evaporated with this apparatus to a final volume
42、of not less than 0,5 ml. If a smaller final volume is required, it is recommended to use a keeper (4.4) in order to avoid significant losses. Decane or dodecane may be used as keepers because they are not detected by the electron- For extracts of samples from clean waters, perform gas chromatographi
43、c analysis at this stage without further clean-up. If the analysis has to be performed with a purification step, proceed to 7.5. Set up the gas chromatograph (5.1), fitted with an electron-capture detector and equipped with a suitable column (5.2) according to the instructions of the manufacturer, a
44、nd ensure it is in a stable condition. Inject the extract (usually between 1 pl and 10 pl but the same volume as that used for calibration) into the gas chromatograph and run a chromatogram. Compare the gas chromatogram obtained to those of the standard solutions (see clause 8). Evaluate the gas chr
45、omatogram qualitatively and quantitatively (see clause 9). The requirements applicable to the extent of the measurements, and the calibration, evaluation and calculation techniques to be used, are described in clause 8. The gas chromatogram obtained is checked for overlapping occurring at the locati
46、ons of the reten- tion times of the determinands of interest. If interfering peaks are present, one of the purification methods de- scribed in 7.5 shall be applied. Otherwise, identify and quantify according to clause 9. 6 Copyright International Organization for Standardization Provided by IHS unde
47、r license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/18/2007 19:40:39 MDTNo reproduction or networking permitted without license from IHS -,-,- 0 IS0 IS0 6468: 1996(E) 7.5 Clean-up and separation Applying the procedure described in 7.2 may lead to coextraction of rela
48、tively polar and/or other undesired substances, which are likely to interfere by the ap- pearance of unknown peaks overlapping the pesticide peaks. NOTE 10 Treatment by column chromatography may help to eliminate some of the substances. However, this method cannot be considered as an absolute system
49、. Use one or both of the following procedures: - clean-up on an alumina-alumina/silver nitrate col- umn, for purification to remove polar compounds (7.5.1); - clean-up on a silica gel column, for separation of PCB from most insecticides (7.5.2). NOTE 11 The quality of each batch of columns should be checked with standard solutions. 7.5.1 Clean-up on alumina-alumina/silver nitrate column C