Assuring comparability of results of nitrate determinations in wastewater: application of metrological principles
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1 Assurig comparability of results of itrate determiatios i wastewater: applicatio of metrological priciples A. Drolc & M. Roš Natioal Istitute of Chemistry, Ljubljaa, Sloveia Abstract I the preset paper, a methodology for the establishmet of traceability ad evaluatio of measuremet ucertaity of results of itrate io determiatio i wastewater is preseted. Nitrate io was determied usig io chromatography (EN ISO , 996). The ecessary relevat iformatio was obtaied from the method validatio data, the quality cotrol data ad equipmet calibratio certificates. The method of measuremet is described together with the measuremet equatio, selected traceable referece stadards ad the associated measuremet ucertaity. The major sources of ucertaity of the results of measuremet were idetified ad the combied ucertaity was calculated. Idetificatio of the mai ucertaity sources represet a basis for target operatio for reducig the measuremet ucertaity of this determiatio. Keywords: itrate, metrology i chemistry, traceability, comparability, measuremet ucertaity. Itroductio Every year may millios of chemical measuremets are made i the EU. It has bee estimated that i the idustrialised coutries measuremets ad related operatios accout 4 to 6 % of the gross atioal product. Measuremets are made for importat purposes, like the determiatio of quality of products, estimatio of the state of the eviromet, implemetatio of evirometal regulatios etc. That makes these measuremets extremely importat. But sadly, all too ofte chemical measuremet results are ureliable. The impact of these poor quality measuremets o the ecoomic ad social live of the EU is eormous. As a cosequece, both govermet ad idustry are icreasigly
2 596 Waste Maagemet ad the Eviromet II recogisig that work is required to improve reliability of chemical measuremets. The beefits will be reduced costs, less risk of major failures ad improved wealth creatio through improved quality of life. The priority that the EC wishes to apply is "low", "medium" ad "high" priority. The high priority ratigs are prevalet for the food ad the agriculture, the eviromet ad for cliical sectors. These ratigs clearly require a correspodig quality of measuremets. Quality of measuremets requires traceability of the results to a stated referece poit (ormaly SI uit), as well as stated rules as the degree of reliability of laboratories results - target values for measuremet ucertaity. Metrology i chemistry leads to iteratioal comparability of results with stated ucertaity of measuremet i order to facilitate the border crossig use of them. Metrology is based o the cocept of traceablity, which is defied as follows: Traceability is the property of the result of a measuremet or the value of a stadard whereby it ca be related to stated refereces, usually atioal or iteratioal stadards, through a ubroke chai of comparisos all havig stated ucertaities. The eed for establishemet of traceability i chemical measuremets is uderscored by the icreasig adoptio of stadards ad measuremet quality systems, such as laboratory accreditatio agaist ISO 7025:999 [3]. Every laboratory eeds to establish traceability ad quatify the measuremet ucertaity by itself for the techiques applied, combiatios matrix/aalyte ad i its ow eviromet [, 2, 3, 4]. The key elemets i the demostratio of measuremet traceability are the followig [5]: () specifyig the measurad ad the acceptable measuremet ucertaity, (2) establishig a suitable method of estimatig the value, (3) validatio of the method, (4) idetifyig the relative importace of each ifluece quatity, (5) choosig ad applyig appropriate referece stadards, ad (6), estimatig the ucertaity. I evirometal sciece developmet of a moitorig system for aalysis of samples is of the highest importace. Cocetratios of pollutats i water samples are measured i order to assess the curret situatio ad represet a basis for decisio-makig ad future sustaiable maagemet of the eviromet. Maagemet strategies are maily based o experimetal data such as chemical aalysis of relevat parameters. I the preset paper, the methodology for establishemet of measuremet traceability ad ucertaity evaluatio is preseted for itrate determiatio i wastewater matrices. Traceability establishmet ad measuremet ucertaity evaluatio are based o ISO ad EURACHEM/CITAC guides [5, 6, 7] ad fulfil the requiremets of the ISO/IEC stadard Experimetal The complete procedure for itrate itroge determiatio i wastewater is stated i the Iteratioal stadard ISO: Water Quality Determiatio of dissolved aios by liquid chromatography of ios Part 2: Determiatio of bromide, chloride, itrate, itrite, orthophosphate ad sulfate i wastewater [8]. Aios are separated usig liquid chromatographic separatio by meas of a separatig colum (Dioex 20, ijectio volume 00 µl). Aio exchager is used as
3 Waste Maagemet ad the Eviromet II 597 statioary phase (guard colum AG4A-SC, separatio colum AS4A-SC) ad aqueous solutios of salts of weak moobasic ad dibasic acids as mobile phases (.8 mm carboate/.7 mm bicarboate, flow of the eluet 2.0 ml/mi). Coductivity detectio is used withi this method (backgroud coductivity 5-20 µs). Coductivity detector is combied with a suppressor device (AMMS-2) which decreases the coductivity of the eluet ad covertes the separated aios ito their correspodig acids. Determiatio of the itrate io is determied by a calibratio of the overall procedure. The calibratio curve is established by measurig a series of itrate referece solutios (approximate cocetratios 0,5 mg/l; mg/l; 2 mg/l; 3 mg/l; 4 mg/l; 5 mg/l; 6 mg/l; 7 mg/l). The cocetratio of itrate, C, is calculated by eq (): A B0 C = () B where A, B ad B 0 are measured area of the sample chromatographic peak, slope of the liear least square calibratio curve ad calculated blak. Slope of the liear least square calibratio curve B ad calculated blak B 0 are calculated from eqs (2), (3), (4) ad (5): ( Ci C ) ( Ai A ) i= B = (2) 2 ( C C ) i= B0 i = A B C (3) A i i= A = (4) C i i= C = (5) where C i ad A i are cocetratio of referece solutio o i th level (C, C i, C ) ad areas of chromatographic peaks of i th referece solutio (A, A i, A ), respectively. 3 Results ad discussio 3. Method validatio The method is a established stadardized method, which has already bee validated. Therefore, oly selected parameters were cosidered to verify the correct implemetatio of the method i the laboratory [9]: trueess ad precisio.
4 598 Waste Maagemet ad the Eviromet II 3.. Trueess Referece material was aalysed (RTC, Aios, I-05, Lot P2) ad o sigificat discrepacy betwee our results ad the certified value was foud. The laboratory also participates i proficiecy tests (AQUACHECK, UK) ad good performace i this determiatio has bee obtaied Precisio The most commo measuremets of precisio are repeatability ad reproducibility. The stadard deviatio of the repeatability of the method was determied to be 0.7%. The stadard deviatio of the itra-laboratory reproducibility was determied by differet aalysts usig the same equipmet over a exteded timescale (three years). It was 2,5 % for samples at the approximate cocetratio level of 0 mg/l. 3.2 Establishig traceability Referece stadards applied i the itrate measuremet procedure are cocetratio of calibratio Certified referece materials (CRM) (see measuremet ucertaity evaluatio, Table ) ad mass Calibratio CRM Traceability of the method was established usig CRM (NIST Stadard Referece Material 385). The certified value of calibratio CRM is based o gravimetric preparatio usig high purity sodium itrate ad io chromatography calibrated usig three idepedetly prepared gravimetric solutios [0] Mass Traceability of mass is provided by regular calibratio procedures for the balace used ad cofirmed by the associated calibratio certificate from a accredited laboratory (traceable to DKD-K-220). The validity is checked o a daily basis with iteral check weights, which are traceable to atioal stadards. 3.3 Measuremet ucertaity evaluatio The mai steps of the measuremet ucertaity evaluatio process are [6]: () descriptio of the method, (2) specificatio of the measurad ad idetificatio of the sources of ucertaity, (3) quatificatio of the ucertaity compoets ad (4) calculatio of combied ad expaded ucertaity Establishig the model The mai sources of ucertaity i the measuremet were idetified as ucertaity of calibratio referece materials, ucertaity of measured areas of referece solutios ad sample ad recovery of the method. Recovery covers possible iterfereces i the method whe samples of differet matrices are aalyzed. With these correctios the cocetratio of itrate (C NO3 ) i a sample was expressed by the model: A B0 CNO 3 = R (6) B
5 Waste Maagemet ad the Eviromet II 599 where R is method recovery ad A is area of the sample. B 0 ad B are calculated accordig to eqs (2), (3), (4) ad (5). The sources of ucertaity i itrate determiatio are schematically preseted i cause ad effects diagram (Figure ) Quatificatio of the ucertaity compoets Ucertaity compoets were determied i oe of the followig ways: experimetally from a series of repeated observatios by calculatig the stadard deviatio (Type A evaluatio), or were obtaied from other sources such as iformatio from calibratio certificates (Type B evaluatio). Before calculatig the combied ucertaity, Type B ucertaities were expressed as oe stadard deviatio. If there were o data o the type of distributio, it was estimated as rectagular or triagular, ad the coverted to a ormal distributio by dividig by factors of 3 or 6, respectively. Ucertaity compoets (see Fig., Table ) are associated with the followig iput quatities Ucertaity associated with liear least squares calibratio The amout of itrate was calculated usig a previously prepared calibratio curve accordig to the liear least squares fittig procedure. The ucertaity of the calculated cocetratio obtaied from the calibratio curve is associated with the ucertaity of the calibratio solutio cocetratios ad the ucertaity of the measured peak areas of the referece solutios. Workig referece solutios were prepared from a stock referece solutio by dilutio. Ucertaity of calibratio CRM was 976 mg/kg ± 6 mg/kg (k=2), as stated i producers certificate (NIST Stadard Referece Material 385). Diluted workig referece solutios were prepared by trasferrig a aliquot of the CRM to a empty, dry, preweighed bottle ad tha reweighig the bottle. From that data exact cocetratio of the workig solutios was calculated. Dilutios prepared gravimetrically eed o correctio for temperature ad o further correctio for true cocetratio i vacuum. The calibratio certificate of the balace idetifies ucertaity sources for tared weighig: the calibratio fuctio of the scale ad the repeatability. The calibratio fuctio has two potetial ucertaity sources, idetified as the sesitivity of the balace ad its liearity. The sesitivity was eglected because the mass was weighed by the same balace over a very arrow rage. The liearity was obtaied from the maufacturer s certificate, where it was reported with a 95% cofidece limit ad coverted to oe stadard deviatio by dividig by a factor of 2, while the repeatability of weighig was obtaied from successive weighig operatios based o data from cotrol charts. Because two successive weighigs are used, ucertaities have to be couted twice. The ucertaities were the combied takig the square root of the sum of the squares of the idividual compoets. Ucertaities of peak areas are maily associated with variatios i flow of the mobile phase. All repeatability cotributios were combied ito oe cotributio for the overall measuremet procedure ad obtaied from the method validatio data.
6 600 Waste Maagemet ad the Eviromet II m Ci Ai C calibr. repeatability m3 calibr. repeatability calibr. repeatability A repeatability m4 m2 liear least square calibratio calibr. repeatability CNO3 RECOVERY Figure : The sources of ucertaity i itrate determiatio - cause ad effects diagram.
7 Waste Maagemet ad the Eviromet II Recovery The method scope covers a rage of sample matrices. Therefore, additioal ucertaity term is required to take accout of differeces i the recovery of a particular sample type. This was evaluated i the laboratory by aalysig a represetative rage of spiked samples, coverig typical matrices (muicipal wastewater, pharmaceutical idustry wastewater, leather idustry wastewater, food idustry wastewater, paper idustry wastewater). The samples were spiked by addig itrate spikig solutio. The sigificace test idicated that the sample recoveries of the measuremets were ot sigificatly differet from, therefore the method was ot corrected for bias. The recovery (R) for each sample was calculated as: Cobs Cative R = (7) Cspike where Cobs is the cocetratio of the aalyte observed for the spiked sample, C spike is the cocetratio of the spike added to the sample ad C ative is the cocetratio of the aalyte i the uspiked sample. The ucertaity associated with recovery was calculated as the stadard deviatio of the recoveries for each sample type aalysed (muicipal wastewater, pharmaceutical idustry wastewater, leather idustry wastewater, food idustry wastewater, paper idustry wastewater) Calculatio of the combied ad the expaded ucertaity Geerally, the result of a measuremet is determied from other quatities ad the relatioship betwee result y ad the values of the iput parameters x i ca be expressed by a model: y = f ( x,x2,...,xi...xn ) (8) The ucertaity of the result (u(y)) depeds o the ucertaity of the iput parameters ad is calculated followig the law of propagatio of errors [8]: N y 2 u ( y ) = u( xi ) (9) xi i= where u(x i ) are the stadard ucertaities of the iput parameters, ad y xi is a sesitivity coefficiet. The sesitivity coefficiet describes how the measuremet result varies with chages i the value of iput estimates. Eq (6) is valid for measuremets where there is o correlatio betwee the iput parameters. The values of the iput parameters C, C 2, C 3, C 4, C 5, C 6, C 7, C 8, A, A 2, A 3, A 4, A 5, A 6, A 7, A 8 ad R of itrate determiatio, as well as their respective stadard ucertaities, the correspodig sesitivity coefficiets ad relative ucertaity variace cotributios, are give i Table. To calculate the expaded ucertaity of the result of a measuremet at the 95% cofidece level, the result for the combied ucertaity was multiplied by a coverage factor of 2. Relative ucertaity variace cotributios are used to illustrate the relative impact of differet ucertaity compoets. The relative cotributio (r i ) of a ucertaity compoet x i to the combied stadard ucertaity is defied here as: 2
8 602 Waste Maagemet ad the Eviromet II Table : Ucertaity compoets of CNO3, their stadard ucertaities, sesitivity coefficiets ad relative variace cotributios. Quatity Symbol Uit Value Stadard ucertaity Sesitivity coefficiet Relative variace cotributio ri, % b) Cocetratio of the solutio C mg/l Cocetratio of the solutio 2 C mg/l Cocetratio of the solutio 3 C mg/l Cocetratio of the solutio 4 C mg/l Cocetratio of the solutio 5 C mg/l Cocetratio of the solutio 6 C mg/l Cocetratio of the solutio 7 C mg/l Cocetratio of the solutio 8 C mg/l Area of the referece solutio A Area of the referece solutio 2 A Area of the referece solutio 3 A Area of the referece solutio 4 A Area of the referece solutio 5 A Area of the referece solutio 6 A Area of the referece solutio 7 A Area of the referece solutio 8 A Area of the sample A Recovery R Nitrate cocetratio CNO3 mg/l a) a) Calculated accordig to eq (9) b) Calculated accordig to eq (0)
9 Waste Maagemet ad the Eviromet II y 2 u( xi ) x i r i = (0) 2 u( y ) where y is the model equatio ((y=f(x, x 2..x i, x N )), x i are iput parameters of the model ad where u(y) is the combied ucertaity calculated accordig to eq (9). The result of itrate determiatio was 5.40 mg/l with a combied stadard ucertaity u(c NO3 ) of mg/l. The measuremet ucertaity was foud to be of acceptable level. Usig a coverage factor of 2, the result of the measuremet should be reported as 5.40 mg/l ± 0.3 mg/l (k=2). The correspodig relative combied stadard ucertaity is 2.5%. The importace of ucertaity sources is dictated by their quatitative effect o the measuremet result. The relative importace of differet ucertaity sources is importat i idetifyig the domiat ucertaity sources with the goal of reducig the overall measuremet ucertaity. The largest cotributios are from the recovery (66.0%) ad repeatability (3.6%), while other cotributios such as cocetratio of calibratio referece materials ad peak areas of calibratio referece solutios are of lower magitude. Therefore, measuremet ucertaity ca be most effectively reduced by icreasig the umber of replicates. 4 Coclusios The establishmet of measuremet traceability ad evaluatio of the ucertaity of a titrimetric method was ivestigated followig EURACHEM/CITAC guides o the traceability ad ucertaity of measuremets. The preseted methodology fulfills requiremets of the stadard ISO/IEC The traceability of results was preseted for routie measuremet ad was accompaied by a measuremet ucertaity evaluatio. Optimized procedure for obtaiig a acceptable estimate of the measurad was established, icludig the calculatio ad a set of measuremet coditios. The method was validated to demostrate that calculatio ad set of coditios are sufficietly complete for the purpose. After that, traceability or cotrol for each value i the equatio ad for each of the specified coditios was established. Traceability was established by calibratio usig a appropriate measuremet stadards. All the data required were obtaied from calibratio certificates, method validatio data ad quality cotrol data. Referece stadards were chose to be appropriate to the equipmet beig calibrated, of appropriate ucertaity ad their values were traceable to relevat refereces. Certificates were issued by a competet authorities. Measuremet ucertaity was evaluated step by step i order to improve uderstadig of the procedure. Detectio of the major ucertaity compoets offers a tool for improvig the performace of the determiatio. Systematic ucertaity budgets, such as the desig preseted here, facilitate the ucertaity evaluatio process ad make it easier to compare cotributios of ucertaity
10 604 Waste Maagemet ad the Eviromet II compoets to the total ucertaity budget, as well as promotig performace improvemet of the method. Ackowledgemets The authors gratefully ackowledge fiacial support from the Miistry of Educatio, Sciece ad Sport of the Republic of Sloveia. Refereces [] Iteratioal stadard ISO: Europea Stadard EN ISO/IEC Geeral requiremets for the competece of testig ad calibratio laboratories, [2] Drolc, A. & Roš, M., Evaluatio of measuremet ucertaity i the determiatio of total phosphorus usig stadardized spectrometric method ISO Acta Chimica Sloveica, 49 (2), pp , [3] Drolc, A., Cotma M. & Roš, M., Ucertaity of chemical oxyge demad determiatio i wastewater samples. Accreditatio ad Quality Assurace, 8, pp , [4] Drolc, A., Roš, M. & Cotma, M., Establishmet of traceability of ammoium itroge determiatio i wastewater. Aalytical ad Bioaalytical Chemistry, 378, pp , [5] Elliso, S.L.R., Kig, B., Rosslei, M., Salit, M. & Williams, A. (eds.). Traceability i Chemical Measuremet, EURACHEM/CITAC, [6] Guide to the Expressio of Ucertaity i Measuremet, BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Iteratioal Orgaizatio for Stadardizatio, Geeve, 995. [7] Williams, A., Elliso, S. L. R. & Rosslei, M. (eds.). Quatifyig Ucertaity i Aalytical Measuremet, EURACHEM/CITAC, [8] Iteratioal stadard ISO: Water Quality Determiatio of dissolved aios by liquid chromatography of ios Part 2: Determiatio of bromide, chloride, itrate, itrite, orthophosphate ad sulfate i wastewater, 996. [9] The Fitess for Purpose of Aalytical Methods, EURACHEM, 998. [0] Natioal Istitute of Stadards ad Techology, Certificate of Aalysis, Stadard Referece Material 385, Nitrate Aio Stadard Solutio.
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