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ColorimetricDeterminationofAmmonium(NH4+)inSolution

ColorimetricDeterminationofAmmonium(NH4+)inSolution
(ver.960129)
ThereareseveralcolorimetricmethodsavailablefordeterminingNH4+concentrationsinwater
samples,soilextractsandplantdigests.ThesemethodsalldetectbothNH4+andNH3formsofN.The
methodwewilluseiscalled"theindophenolbluemethod"or"phenatemethod."Itisbasedonthe
reactionofNH3 inalkalinesolutionwithphenatetoproduceabluecolor(indophenolblue)inthe
presenceofastrongoxidizingagent,suchashypochlorite.Althoughthereactionwillproceedatroom
temperature,itcanbespedupbyheatingthesolution(35oC)and/orusingametalcontainingcatalyst
suchassodiumnitroferricyanide(nitroprusside).Manymodificationsofthismethodareavailable.
TheonewewilluseisbasedonSolorzano(LimnolOceanogr.1969.14:799801),modifiedfora10
mlsamplevolume.
OthermodificationsofthismethodcanbefoundinMethodsofSoilAnalysis(Pageetal.,eds.,
AmericanSocietyofAgronomy,Inc.andSoilScienceSocietyofAmerica,Inc.,Madison,WI),
MethodsofSeawaterAnalysis(VerlagChemienotinourlibraryandmaybehardtofind)and
StandardMethodsfortheExaminationofWaterandWastewater.(Clescerietal.,eds.,American
PublicHealthAssociation,Washington,DC).
Thephenatemethodalsohasbeenadaptedforuseonvariousautomatedanalyzers,suchasthose
manufacturedbyTechnicon,AlpkemandLachat.Seethemanualforthespecificmachineyouare
using.
Notes:
Usetesttubeswhichcanaccommodatea10mlsample.Allglasswareshouldbeveryclean(scrubbed
withhotwater,rinsedwith0.1NHCL,rinsed6timeswithpurifiedwater,andrinsedoncewith
reagentgradedeionizedwater).Besurewaterforreagentsandstandardsisammoniafree.Ordinary
distilledwatermaycontainammoniumusefreshlydeionizedwater.
Ammoniaisvolatileandispresentintheair.Manydifferentsourcesofammoniacancontaminatethe
samples.Smoking,ammoniadetergents,freshlycutgrass,openingabottleofammoniumhydroxide
inthelaboratory,etc.Exercisecaution.
Veryturbidsamplemayneedtofilteredorcentrifugedpriortoanalysis.Precipitationofcalciumand
magnesiumhydroxidecaninterferewiththeanalysis.Thiscanbepreventedbyadditionofa
complexingreagentsuchascitrateorEDTA.SeeMethodsofSoilAnalysisforfurtherdetails(Page,
A.L.,R.H.MillerandD.R.Keeney,eds.1982.MethodsofSoilAnalysis.AmericanSocietyof
Agronomy,Inc.,Madison,WI)
StockReagents:
1)Phenolalcoholreagent:dissolveadd10gofphenolin95%ethylalcoholtoafinalvolumeof100
ml(toxichandlewithcare).
2)Sodiumnitroprusside(nitroferricyanide):dissolve1ginDIwatertoafinalvolumeof200ml.
Storeindarkbottlefornotmorethan1month.(toxic)
3)Alkalinecomplexingreagent:dissolve100goftrisodiumcitrateand5gofsodiumhydroxideinDI
watertoafinalvolumeof500ml.
4)Sodiumhypochlorite:usecommercialbleach(i.e.,Chlorox),asnewaspossible.
5)Oxidizingsolution:add100mlalkalinesolution(3)to25mlsodiumhypochlorite(4).Prepare
freshdaily.
Calibrants:
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ColorimetricDeterminationofAmmonium(NH4+)inSolution

StockSolutionA1000ppmNH4Nstock.Dissolve4.7168gofdry(110oCfor12h)(NH4)2SO4 in
approximately900mlofDIwaterina1Lvolummetricflask.Bringto1LwithDIwater.Canbe
preservedbyaddingtwodropsofchloroform.Labelwiththesolution,thedateandyourinitials.
StockSolutionB100ppmNH4Nstock.Transfer10mlofStocksolutionAtoa100mlvolummetric
flask.DilutetomarkwithDIwater.Labelwiththesolution,thedateandyourinitials.
Workingstandards(canbeadjustedtocovertherangeofsamplesbeinganalyzed).Using100ml
volummetricflasks,preparethefollowingseriesofworkingstandards.
VolumeofStockBVol.ofstandardConcentration
1ml100ml1000gNH4N/L
750l100ml750gNH4N/L
500ml100ml500gNH4N/L
200ml100ml200gNH4N/L
100ml100ml100gNH4N/L
50ml100ml50gNH4N/L
0ml100ml0gNH4N/L
Assay:
1.Add10mlofstandardorsampletolabeledtesttubes.
2.Toalltubesadd0.4ml(400l)ofphenolsolution(1),0.4mlnitroferricyanide(2)and1ml
oxidizingreagent(5).(Notevolumescanbeadjustedaccordingtosamplesize)
3.Mixwell(invertscrewcaptubes).Letdevelopforatleast1h(3hisbetter)indarkness.Mix
samplesperiodicallywhiledevelopmentisoccurring.
4.Readabsorbanceofstandardsandunknownsamplesusingaspectrophotometersetto630
nm.
5.Useabsorbancevaluesofstandardstogenerateastandardcurve.Calculateconcentrationsin
unknownsfromstandardcurve.
6.DisposeofallassaywasteintoalabeledbottleDONOTPUTPHENOLDOWNTHE
SINK!
Limitations:
Thedetectionlimitisreportedtobeabout10gNH4N/LandBeer'slawisobeyeduptoabout
500gNH4N/L.Wehavefoundthatthemethodislineartoatleast750gNH4N/L,and
Koroleff(1976)reportsthathigherconcentrations,upto1200gNH4N/L,canbemeasured
withaonecmflowcellandanappropriatestandardcurve.
Samplepreparationandstorage:
Turbidsamplesandsoilextractswillneedtobefilteredorcentrifugedpriortoanalysis.If
filtrationisused,bewareofammoniumcontaminatedfilters(Whatman42isnotacceptable).
WehavehadsuccesswithPoreticspolycarbonatemembranefilters(0.4poresize).
Samplesshouldbestoredinglassorpolyethylene,andkeptrefrigerateduntilanalyzed.Ammoniumis
verylabileandsamplesshouldbeanalyzedassoonaspossible(preferablywithin7days).Donot
freezesamplesbeforeanalysis.Ifnecessary,samplesmaybepreservedbyadditionof0.8mlconc.
H2SO4topH<2andthenrefrigerated.However,samplesmustthenbeneutralizedbeforeanalysis,and
contaminationbyabsorptionofatmosphericNH3maybeaproblem..
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SampleswithconcentrationsabovethehighstandardshouldbedilutedwithDIwater(orother
appropriatematrix,dependingonthenatureofthesamples)beforeadditionofreagents.
References:
Koroleff,F.1976.Determinationofammonia.InMethodsofSeawaterAnalysis(K.Grasshoft,ed.).
VerlagChemie,pp.126133.
BIOL826Spring1997
LabReport#1
Thefirstlabreportwillcoverthefirstthreeweeksoflabs,andwillbeaboutcolorimetricanalysisof
solutionsamplesforphosphate,ammoniumandnitrate.Itwillnotbedueuntiltheweekafterwe
completeallthreelabs(dueFebruary18).Keeptrackofyourdatafromthesethreelabs.Specifically,
youwillneed:
1.recordsoftheconcentrationsandabsorbancesofyourstandardsandsamples.
2.agraphicpresentationofyourstandardcurveforeachanalysis.
3.theregressionequationsrelatingabsorbancetoconcentration.
4.calculatedresultsforyourunknowns.
5.descriptionsofmethodology,includinganymodificationstotheprocedures,orproblems
encountered.
Yourlabreportshouldinclude:
Introduction:Whyareweinterestedinmeasuringnutrientconcentrationsinsolution?forwhatkinds
ofsamplescantheseanalysesbeused?whatarethegeneralprinciplesbehindallofthese
measurements?
Methods:principlebehindthereactionusedlimitationsbriefdescriptionofthemethodrangeof
standardsused.
Results:presentationofstandardcurvesanalysisofunknownsanymysteriousdevelopments
Discussion:potentialsourcesoferrorexplanationsofanomalousresultssuggestedwaystoimprove
theaccuracyofthemeasurements.
References:sourcesfordetailedinformationonthesemethods,sothatyouwillhavereferencesforthe
future.

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