Standard Operating Procedures for Measuring Bulk Stable ...
Transcript of Standard Operating Procedures for Measuring Bulk Stable ...
NOAA Processed Report NMFS-NWFSC-PR-2020-04
https://doi.org/10.25923/3mwp-ce02
Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS)
February 2020
U.S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric AdministrationNational Marine Fisheries ServiceNorthwest Fisheries Science Center
NOAA Processed Report Series NMFS-NWFSC-PR
The Northwest Fisheries Science Center of NOAA’s National Marine Fisheries Service uses the NOAA Processed Report NMFS-NWFSC-PR series to disseminate information only. Manuscripts have not been peer-reviewed and may be unedited. Documents within this series represent sound professional work, but do not constitute formal publications. They should only be footnoted as a source of information, and may not be cited as formal scientific literature. The data and any conclusions herein are provisional, and may be formally published elsewhere after appropriate review, augmentation, and editing.
NWFSC Processed Reports are available from the NOAA Institutional Repository, https://repository.library.noaa.gov.
Mention throughout this document of trade names or commercial companies is for identification purposes only and does not imply endorsement by the National Marine Fisheries Service, NOAA.
Cover image: Reactor reagents used for bulk carbon and nitrogen analysis. Clockwise from the top: silvered cobaltous, reduced copper, chromium oxide. Also depicted is an outline of nitrogen and carbon dioxide peaks; from left to right: N2 sample peak, CO2 sample peak, and two CO2 reference gas peaks. Photograph by J. Gates, NMFS/NWFSC.
Recommended citation:
(Gates et al. 2020)1
1 Gates, J. B., P. M. Chittaro, and K. B. Veggerby. 2020. Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS). U.S. Department of Commerce, NOAA Processed Report NMFS-NWFSC-PR-2020-04.
https://doi.org/10.25923/3mwp-ce02
U.S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric AdministrationNational Marine Fisheries ServiceNorthwest Fisheries Science Center
Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS)Jonelle B. Gates,1 Paul M. Chittaro,1 and Karl B. Veggerby2
https://doi.org/10.25923/3mwp-ce02
February 2020
Environmental and Fisheries Sciences DivisionNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, Washington 98112
Fish Ecology DivisionNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, Washington 98112
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Contents
1.0 Introduction...................................................................................................................................................................... 1
1.1 ScopeandApplicability................................................................................................................................... 2
1.2 SummaryofProcedure.................................................................................................................................... 2
1.3 Definitions............................................................................................................................................................. 2
1.4 Safety....................................................................................................................................................................... 2
2.0 DocumentationandRecordkeeping...................................................................................................................... 3
3.0 EquipmentList................................................................................................................................................................ 3
3.1 Materials,PerSample....................................................................................................................................... 3
3.2 SolventsandCleansers.................................................................................................................................... 3
3.3 OtherMaterialsandApparatus.................................................................................................................... 4
4.0 SamplePreparation/Dissection............................................................................................................................. 4
4.1 LabelingSampleVials...................................................................................................................................... 4
4.2 PreparingDissectionEquipment(ifapplicable).................................................................................. 5
4.3 DissectingTissuefromaSingleSpecimen.............................................................................................. 5
4.4 SubsamplingfromaHomogenizedSampleorSampleComposite............................................... 5
4.5 CleaningtheDirtySpatulas........................................................................................................................... 6
5.0 FreezeDrying.................................................................................................................................................................. 6
5.1 PreparingtheFreezeDryer........................................................................................................................... 7
5.2 LoadingtheSamplesintotheFreezeDryerChamber....................................................................... 7
5.3 RemovingtheSamplesfromtheFreezeDryer...................................................................................... 7
6.0 LipidExtractionontheAutomatedSolventExtractor(ASE)...................................................................... 8
6.1 PrepareASECells.............................................................................................................................................. 8
6.2 PrepareSamples................................................................................................................................................ 8
6.3 Loading/OperatingtheASE.......................................................................................................................... 9
6.4 UnloadingSamplesfromtheASEPost-LipidExtraction.................................................................. 9
6.5 CleaningtheASECells.................................................................................................................................... 10
7.0 Homogenization........................................................................................................................................................... 10
7.1 HomogenizingSamplesPost-LipidExtraction..................................................................................... 11
7.2 HomogenizingSampleswithoutLipidExtraction.............................................................................. 11
7.3 CleaningtheCapsules,Spatulas,andForceps......................................................................................12
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8.0 WeighingSamples.........................................................................................................................................................13
8.1 BuildingaLoadingSheet...............................................................................................................................13
8.2 PillingofSamplesforAnalysis....................................................................................................................13
8.3 FinalizingtheLoadingSheet........................................................................................................................15
9.0 LoadingtheSamplesontheElementalAnalyzer(EA).................................................................................15
9.1 InstrumentMaintenance...............................................................................................................................15
9.2 RunningLinearityandOn/Offs.................................................................................................................. 16
9.3 CheckingtheOn/OffsandLinearityChecks......................................................................................... 16
9.4 CheckingtheBackgroundintheIRMS.................................................................................................... 18
9.5 BuildingaSequencein Isodat ..........................................................................................................18
9.6 LoadingtheEAAutosampler...................................................................................................................... 19
9.7 StartingtheRun................................................................................................................................................ 19
9.8 Post-Run............................................................................................................................................................... 19
10.0 ExcessSampleArchival.............................................................................................................................................20
11.0 ProcessingtheData.....................................................................................................................................................20
11.1 ManipulatingtheRawData.........................................................................................................................20
11.2 ProcessingtheDatainR ....................................................................................................................20
12.0 Data.....................................................................................................................................................................................21
12.1 DataReview.........................................................................................................................................................21
12.2 DataStorage........................................................................................................................................................21
13.0 QualityAssurance.........................................................................................................................................................21
13.1 QuantitationRange..........................................................................................................................................21
13.2 InstrumentCalibration.................................................................................................................................. 22
13.3 ContinuingCalibrationVerification(CCV)............................................................................................ 22
13.4 ReferenceMaterials........................................................................................................................................ 23
13.5 MethodBlanks................................................................................................................................................... 23
13.6 SampleReplicates............................................................................................................................................24
13.7 ReportedResults..............................................................................................................................................24
References................................................................................................................................................................................... 25
AppendixA:InstructionsforSubmissionofSamplesforStableIsotopesAnalyses...................................26
AppendixB:SubmissionFormsforStableIsotopesAnalyses..............................................................................30
ProjectInformation.....................................................................................................................................................30
SampleInformation.....................................................................................................................................................31
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1.0 Introduction
Theisotoperatiomassspectrometer(IRMS)housedattheNorthwestFisheriesScienceCenter(NWFSC)isusedtoconductaccurateandprecisedeterminationsofisotoperatiosofcarbonandnitrogen.NWFSC’sEnvironmentalChemistryProgramlabfacilityincludesaThermoFisherScientific’sFlash2000ElementalAnalyzercoupledwiththeConfloIVinterfaceandtheDeltaVAdvantageIRMS.Thesesystemsarecomplexandsensitiveandrequiredetailedcareandregularmaintenancetoensureahighlevelofperformance.Thisprocessedreportiswrittentobeusedinconjunctionwithhands-ontraining.Itismeanttobetreatedasalivingdocumentandwillbeupdatedaschangesaremadeintheprocedures.
Thisreportprovidesstep-by-stepinstructionsforsamplepreparation,instrumentoperation,maintenanceoftheinstruments,samplearchival,qualityassurance(QA)andqualitycontrol(QC)measuresandcriteria,anddatareporting.TheQuality Assurance Plan for Analyses of Environmental Samples(Sloanetal.2019)wasreferencedthroughoutSection13.Referenceinformationcanbefoundattheendofthisreport.
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1.1 Scope and Applicability
Thismethodwasdevelopedusingapproximately1gramofmarinebiotatissueandproducesahomogenized,stablesamplethatcanbestoredatroomtemperatureindefinitely.Thishomogenatecanbesampledmultipletimesforbulkcarbonandnitrogenstableisotopeanalysisaswellascompound-specific15Nanalysisofaminoacids.
Ittakesapproximately3–7daystoprocessasamplesetsubmittedforbulkstableisotopeanalysis,dependingonsamplesetsizeandwhetherornotthesethastobelipidextracted.Ittakesabout8minutespersampletorunontheEA-IRMSforbulkcarbonandnitrogenstableisotoperatios.
1.2 Summary of Procedure
1. Tissuesamplesaresubsampledandplacedinalabeledscintillationvialwithcap.Samplesareplacedina–80°Cfreezerforatleastanhouroruntiltheyarefrozensolid,andthenplacedinafreezedryerfor24hourstoremovewater.
2. Iflipidremovalisrequired,thensamplesareloadedontoanacceleratedsolventextractor(ASE)350toobtainlipid-freesamples.Formorein-depthdetailsoftheASE350extractionmethod,seeHermanetal.2005.
3. Samplesthatdonotrequirelipidremovalorpost-lipidremovalsamples,arehomogenizedusingaMixerMill(SPEXSamplePrep5100).
4. Homogenizedsamplesandstandardsarethenweighedintoindividualtincapsules,andthesearerunontheIRMS.
5. DatafromtheIRMSarerunthroughastandardQA/QCanddataprocessingprocedure,andfinalizeddataarereported.
1.3 Definitions
ASE: acceleratedsolventextractor IRM: internalreferencematerialCSIA:compound-specificisotopeanalysis IRMS:isotoperatiomassspectrometerDQO:dataqualityobjective SOP:standardoperatingprocedureEA:elementalanalyzer SRM:standardreferencematerial
1.4 Safety
Thismethodusesnumerouschemicalsandreagentsthatareconsideredhazardousforflammabilityandtoxicity.Therefore,afumehoodisusedwhenworkingwithhazardousmaterialstominimizeexposureoftheanalysttosolventvapors.Whenworkingwithallchemicals,aminimumofnitrileglovesandeyeprotectionareworn.AdditionallaboratorysafetypracticesarefollowedaccordingtotheNWFSCChemical Hygiene PlanandtheNWFSCChemical Waste Management Guide.TheManageMaterialSafetyDataSheets(MSDS)shouldbereviewedforallchemicalsandreagentspriortostartingthisprocedure.
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2.0 Documentation and Recordkeeping
Itisimperativethatallstepsthroughouttheprocessprocedure(e.g.,samplepreparation,maintenanceoftheinstrumentation,andoperationoftheinstrumentation)arerecordedintheappropriatelogbookswithcorrespondingsamplesetnumber,dates,andsamplepreparer/instrumentoperatorinitials.ItisalsoimportanttoupdatethestatusofthesamplesetinEnvironmentalChemistry’sGoogleDriveunderSI Current Set Status.
3.0 Equipment List
Itiscrucialthatallscalpels,spatulas,forceps,pulverizationchambers,andsteelbearingsarecleanedwithoil-removingdetergent(Micro-90detergent),anappropriatescrubbrush,amplewaterrinses,followedbysonicationinanappropriatesolvent.Thesestepswillhelppreventpotentialcontaminationthatcouldaffecttheisotopemeasurements.
3.1 Materials, Per Sample
Non-LipidExtractionSamples• 1ea.20-mLscintillationvialwithpolyethylenelinedcap• 1ea.cleanstainless-steelpulverizationchamberwithbearing• 1ea.4×6tincapsule
LipidExtractionSamples• 2ea.20-mLscintillationvialwithpolyethylenelinedcap• 5.5-cmroundglass-fiberfilter—VWR696• 250-mLAcceleratedSolventExtractor(ASE)collectionbottle• 3glass-fiberfiltersperASEcell,firedovernightat400°Cinamufflefurnace• 1cleanASEcellpersample(seeSection6.5)• 1ea.4×6tincapsule
3.2 Solvents and Cleansers
• methylenechloride(forASE)• acetone(forcleaningsuppliesviasonication)• Micro-90detergent
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3.3 Other Materials and Apparatus
• AcceleratedSolventExtractorASE350—DionexCorpASE• desiccator• VirtisFreezemobile12XL• MixerMill—SPEXSamplePrep5100• ultrasoniccleaner—Branson5800• stainlesssteelscalpelwithblades• stainlesssteelspatulas• forceps• paperclips,rinsedwithacetone• 96-welltrays• six-placebalance—MettlerToledoExcellencePlus• two-placebalance—AdventurerOhausAR3130• –20°Cfreezerfortemporarysamplestorage• –80°Cfreezerforlong-termsamplestorageandpre-freezedry• pillingtools• frozeniceblock• DeltaVAdvantageIRMS• ThermoScientificFlash2000OrganicElementalAnalyzerFlashHTPlus• ThermoScientificConfloIV
4.0 Sample Preparation/Dissection
Priortosamplepreparation,refertotheInstructionsforSubmissionofSamplesforStableIsotopesAnalyses(AppendixA)aswellastheSubmissionFormforStableIsotopesAnalyses(AppendixB).Oncethesamplesethasbeenscheduled,itisbesttolocatethesamplesandensuretheyarereadytobesubsampled.Ifthesamplesneedtobehomogenized,doso.Makesuretokeepallpaperworkthatisassociatedwiththesamplesettogetherinthecorrespondingfolder.
4.1 Labeling Sample Vials
Beginbypreparingclothlabelsfortheentiresetbywritingtheassignedlabnumber,fieldID,andthesetnumberonthelabelforeachsample(setnumberisfoundonwhitesheet).Attacheachlabeltoanewglassscintillationvialandrecordthelabnumberontheviallidusingafeltpen.Ifthesetrequireslipidextraction,theinitialscintillationvialonlyrequiresthelabnumberwrittenwithafeltpenonthecapandvial.Post-lipidextraction,thesamplewillbetransferredtoanewscintillationviallabeledwithclothlabelsthatincludethelabnumber,fieldID,andthesetnumberonboththecapandvial.
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4.2 Preparing Dissection Equipment (if applicable)
Cutaluminumfoilsquaresforeachsample.Inafumehood,rinsethefoilsurfacewithacetoneandletitdry.Pullthefrozeniceblockfromthefreezerandplaceinthehood.Covertheworkingsurfaceoftheblockwithnewacetone-rinsedaluminumfoil.Afterassemblingthebladesontothescalpelhandles,acetone-rinsethebladesinafumehoodandsetthemonacleanpieceofaluminumfoil.PlaceaQuickSmartautomaticscalpelbladeremovernexttoyourdissectionsurface.Locatecleanforcepsandplacethemnearyourworkspace.
4.3 Dissecting Tissue from a Single Specimen
Removethetissuefromthesamplecontainerusingforcepsandplaceitonthecleanaluminumfoilatopoftheiceblock.Fortheskinanalysis,carefullycuttheskinfromthefieldsampleusingacleanscalpel,beingcarefultoremovealloftheblubberandmuscle.Forblubberanalysis,carefullycuttheblubberfromthefieldsampleusingascalpel,beingcarefultoremovealloftheskin.Forallothertissues,removeasmallpiecefromthemiddlesection,toavoidtheareasofthesamplethatmighthavebeenexposedtofreezerfrost.Cutthesampleintosmallpieces(~0.1gorsmaller)usingthescalpelandplacethemseparatelyinthelabeledscintillationvial,placingthembackinthefreezerbetweeneachsample.
4.4 Subsampling from a Homogenized Sample or Sample Composite
Placeallofthesamplejarsouttothawforatleast45minutespriortosubsampling.Afterthesampleshavethawed,scoopoutasmallamountofthesampleusingacleanspatula,tryingtoavoidtheverytoplayer.Useanew,cleanspatulaforeachsample.Placethelabeled20-mLscintillationvialonthetwo-placebalanceandweighoutasmallamountofthesample.Theweightsdonotneedtoberecordedandareonlyusedtomakesureenoughsamplehasbeensubsampledandthatsamplematerialisnotwasted.Approximatesubsamplemassesarelistedbelow:
• Forwholebodyfishsamples,itisbesttoweighoutatleast0.5goftissue,keepinginmindthattheremightbepiecesofskinorboneinthesubsamplethatcannotbegroundtoapowder.
• Forfishmusclesamples,subsample0.2–0.4goftissue.• Formusselorshellfishsamples,weighoutatleasta1-gsampleduetothe
increasedamountofwaterineachsamplecomparedtoblubberorfishmuscle.
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4.5 Cleaning the Dirty Spatulas
Aftercompletingthesubsamplingstep,takethedirtyspatulastothesinkandscruboffanyresidualtissueusingMicro-90soap.Itisbesttoworkwithgloves,asthedetergentisconcentratedandcorrosive.Oncethespatulasarecleanoftissue,placethemintheperforatedstainlesssteelinsertinsideofthesonicatorandfillwithwaterandMicro-90detergenttothefillline.Neveroperatethesonicatorwithoutaninsert.
Afterthesonicationstepiscompleted,drainthewaterfromtheunitusingthevalvelocatedontheleftside.Openthevalveandletthewaterflowdownthedrainlocatedinthefumehood.Removetheinsertandrinseeverythingunderwater.Laythespatulasoutonabsorbentpapernexttothesinktodryovernight.
Whenthespatulashavecompletelydried,placetheminthesolidsonicatortrayinsertandsubmergewithacetoneinafumehood.Makesuretousetheappropriatenitrilegloveswhileworkingwiththeacetone.Fillthesonicatorwith1,650mLofwaterbeforeinsertingthesolidtrayinsertintotheunit.Settheunittosonicatefor30minutes.
Afterthesonicationstepiscompleted,drainthewaterfromtheunitusingthevalvelocatedontheleftsideoftheunit.Openthevalveandletthewaterflowdownthedrainlocatedinthefumehood.Wearingnitrilegloves,removethesolidtrayinsertanddraintheacetoneintoawastebucket.Pourtheacetonewasteinthewastecontainerlabeled100% acetone,locatedinthefumehood.Leavetheinserttositinthefumehooduntilallofthesolventhascompletelyevaporated(atleast30minutes).Returnthecleanspatulasfromtheinsert,usinggloves,totheglassjarlabeledSpatulas.
5.0 Freeze Drying
Afterallofthesampleshavebeensubsampled,loosenallofthecapsofthesamplesandplacethemina–80°Cfreezerforatleastanhourpriortofreezedrying.
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Open. Closed.
5.1 Preparing the Freeze Dryer
Beginbycheckingtheleveloftheoilintheroughpumptomakesurethattherewerenoleaksbetweenruns.Theroughpumpcanbeaccessedbyopeningthedoorlocatedtotherightofthecondensingunit.Checktheleveloftheoilinthesightglassandactaccordingly.IftheoillevelisOKtooperate,latchtherefrigeratordoorallthewayshut(itshouldbeopenfromthelastrunsothatthechambercandryout).UsingtheRefrigerateswitch,turntherefrigeratoronandletitcoolforthehourthatyoursamplesareinthe–80°Cfreezer(untilthefreezedryerreachesatleast–30°C).
5.2 Loading the Samples into the Freeze Dryer Chamber
Removethelidtothechamberandplaceitonitssideonthetable.Removethetrayfrominsideofthechambersothatthesamplescanbeplacedontheshelveseasily.Placethesamplevialsonthefreezedryertraysandbesurethecapshavebeenslightlyloosenedsothatmoisturecanescape.Onlyplacesamplesonthemiddleandbottomshelves(notontopoftherack).Duringthevacuumchange,samplesmaycomeoutoftheirvialsandcontaminatenearbysamples,soitisadvisabletokeepthecapsonbutloosened.
Next,placethevacuumchamberlidon,makingsureitiscenteredaroundthelipofthechamber,andclosethewhitevalvelocatedonthetopofthelid.Thevalveisclosedwhenthehandleisinthebackandthesmalldimpleisvisibleinthevalvewindow.SwitchonthevacuumusingtheswitchlabeledVacuum.Oncethefreezedryerissealed,startthevacuumpump.Afterabout15minutesitisbesttocheckbackandmakesurethatthevacuumsensorisreadingalowvacuum(approximately15Millitorr).Forsufficientsamplelyophilization(waterremoval),lettheunitrunforapproximately24hours.
Recordthesamplesetinformation,thedate,yourinitials,thematrixaswellasthenumberofsamplesinthefreezedryerinthefreezedryerlogbook.BesuretoupdatethefileentitledSI Current Set Status,whichisfoundinEnvironmentalChemistry’sGoogleDrive.
5.3 Removing the Samples from the Freeze Dryer
Whenthesamplesaredry,stopthevacuumpumpbyswitchingtheVacuumswitchoff.SwitchoffRefrigerateaswell.Slowlyopenthewhitevalvelocatedatopthevacuumchamberlidandlettheairout—slowly,sothattheairsurgedoesnotdisruptthesamples.Oncethereisnolongervacuum,removethechamberlid,settingitonitsside,andremovethetrayfrominsidethechamber.Removethesamples,tightenthecaps,andplacetheminorderinthevialtray.
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Leavethecondenserdoorcrackedopen,withafewpapertowelsinthechambertohelpspeedupthedryingprocess.ExaminetheO-ringslocatedonthecondenserdoorandvacuumchamberforanytearsordebris,andmakenoteiftheyneedtobelubricatedorreplaced.Placethevacuumchamberlidbackontopoftheunitsothatdustanddebrisdonotcollectintheunitwhileidle.
If the samples do not require lipid extraction, skip Sections 6 and 7. Proceed to Section 8.
6.0 Lipid Extraction on the Automated Solvent Extractor (ASE)
TheASEcellsthatareusedtolipid-extractsamplestoberunforstableisotopeanalysisarekeptseparatefromtheothers(theyareonlysonicatedinAcetonebetweenruns).
6.1 Prepare ASE Cells
SetoutallofthecleanASEcellsthatyouplantouseand,usingafeltpen,labelthebodyofeachcellwiththesamplenumber.Unscrewthetopcapofthecelland,usingforceps,placetwofiredglass-fiberfiltersinside.Usingtheappropriateblackrod,pushthefiltersdowntothebottomofthecell.Makesurethatthebottomcapisontight.
6.2 Prepare Samples
Wearinggloves,takea5.5-cmdiameterglass-fiberfilter,folditinhalftwice,andopenonesidetomakeafiltercone.Becarefulwhenhandlingtheglass-fiberfilters;theyarefragile.Clipthreeofthefoldedsidesofthefilterconetogetherwithacleanpaperclipandattachthecliptotheedgeofaclean,labeledASEcellinordertoholdtheconeopen.
Usingacleanspatula,scoopthesampleoutofthevialandplaceitinthefiltercone.Becausethesamplesaredry,youcanuseonespatulaforthewholeset,makingsuretowipeitoffbetweensampleswithapapertowel.Wearinggloves,foldtheconeinhalfandpapercliptheconeclosed.Placethesamplepocketinsideoftheappropriatelabeledcell.Usingforceps,putoneglass-fiberfilterontopofthesampleandpushitdowngentlywiththeblackrod.PlacethecellcapontightlyandloadthesampleontheASE.Repeatthisprocessuntilallofthecellshavebeenused.
Ifthereistoomuchsampletofitintoonefiltercone,anotherconeshouldbemadeortherestofthesampleshouldbediscarded.Thevialthatthesamplewasfreeze-driedinwillbediscardedintheBroken GlassboxafterthesamplehasbeenloadedintheASEcell.
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MostSIsetscontaintoomanysamplestolipid-extractinasinglerunontheASE,sotheywillrequiremultiplerunsontheASEandpropercleaningbetweensamplesets.SeeSection6.5forhowtocleantheASEcells.
6.3 Loading/Operating the ASE
Beginbyopeningthevalveonthenitrogentanksothatthesystemhasgasflow.LoadallofthecellsonthetopcarouseloftheASEinorderofsamplenumber.Makesurethereisawastebottle/vialforeachcellthathasbeenloadedaswellasarinsebottle/vialintraypositionsR1andR2.Removethesolventreservoir,placingthespargeinacleanASEvialtemporarily,andcapthebottle.Workinginthehoodwiththeappropriatenitrilegloves,fillthesolventreservoirwithmethylenechloride.Hookthesolventreservoirbackuptotheinstrument.
Usingthekeypadontheinstrument,locateMethod 3,themethodusedforlipidextraction.Theparametersshouldmatch:
Cycles:2Purge:60secondsStatic:5minutesHeat:0minutesRinseVolume:80%Temperature:0°CCellType:SSTSolventSaver:OffSolventA:1MethyleneChloride
Double-checkthatyouhavethesamenumberofcellsaswastebottles,aswellasrinsebottlesintraypositionsR1andR2.PresstheRinsebuttontorunamanualrinse,andrepeatthatsteptwomoretimes.Afterthesystemhasbeenthoroughlyrinsed,presstheStartbuttonandallowthesystemtolipid-extractthesampleset.
6.4 Unloading Samples from the ASE Post-Lipid Extraction
Afterallofthesampleshavebeenlipid-extracted,presstheTraybuttonuntilthegreenindicatorlightmovestotheleftsideofthebuttonwhichreleasesthesampletrays.Workingwithnitrilegloves,removeallofthewastebottlesandpourthemethylenechlorideintothelabeledmethylenechloridewastebottlestoredinafumehood.Tosstheperforatedcapsawayandcompletelydrythebottlesinthefumehoodforseveralhoursbeforerecappingthemforthenextset.Thebottlesarereusedforlipidextractingstableisotopesamplesorrinsingcells.Asnotedpreviously,itmaytakemorethanoneroundofASEextractionstolipid-extractanentireSIsampleset.
RemovetheASEcellsfromtheuppertrayandplacetheminafumehood.Removethecellcapand,usingforceps,removethesamplefromthecellandplaceitinanewlylabeledscintillationvial.Looselyplacethecaponthescintillationvialsothatthesolventcan
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fullyevaporatefromthesample.Repeatthesestepsuntilallsampleshavebeenremovedfromthecells.Oncethesolventhasevaporatedfromthesamples,tightenthecapsandremovethemfromunderthefumehood.Leavethefiltersinthehooduntilthesolventhasevaporatedfromthemandthendisposeofthem.
6.5 Cleaning the ASE Cells
AftertheASEcellshavebeendismantled,placeallofthecapsinacylindricalsteelsonicatortub.Workingwithnitrilegloves,removethesamplenumbersfromthecellbodieswithacetoneandapapertowelandplacethebodiesinanothercylindricalsteelsonicatortub.Fillbothtubswithacetoneuntilallofthecapsandbodiesaresubmerged.Placethetubsinsidetheperforatedsonicatorinserttrayandfillthetrayaroundthetubswithwatertothefillline.SonicatetheASEcellpartsfor30minutes.Afterthesonicatorhasfinished,pourtheacetoneintotheappropriatewastecontainerandtransferittotheflammablewastecontainerunderthefumehood.Layallofthecellpartsoutonacleanpieceoftinfoiltodryinthefumehood.Oncethesolventhasfullyevaporatedfromthecellparts,reassemblethecellsandstorethemintheirassignedspace.
7.0 Homogenization
Inordertoachievesamplehomogeneity,samplesmustberunthroughaMixerMillandgroundforapproximatelythreeminutesusingasteelballbearinginasteelcapsule.
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7.1 Homogenizing Samples Post-Lipid Extraction
Beginbycarefullyopeningthebundlewithcleanforcepsorcleangloves.Unfoldthefilterconeand,usingacleanspatula,transferthedriedsampleintoacleanpulverizationcapsuleequippedwithasteelballbearing.Placethelidontothecapsulebody,makingsurethatitisequippedwithanO-ring.ClampthechamberintotheMixerMill,makingsurethatthereisrubbercushioningoneithersideofthecapsuleinsideoftheclamp.Setthetimerfor3minutesandpushthewhitebuttonlocatedinthemiddleofthetimerdialtopulverizethesample.
WhentheMixerMillhasfinished,removethechamberfromtheMixerMillandremovethelidfromthebodyofthecapsule.Ifthelidhasbecomestucktothebody,lightlytapthecapsuleonitssidesandtryremovingthelidagain.Repeatasmanytimesasittakestoremovethelid.Usingacleanspatula,transferthepulverizedsamplefromthecapsuleandlidbackintothelabeledscintillationvial.Repeatthisprocessforeachsample.
SomesamplesetsmaybelargerthanthenumberofMixerMillcapsuleswecurrentlyhave,sobesuretoproperlycleanthecapsulesbetweenhomogenizationevents.SeeSection7.3.
7.2 Homogenizing Samples without Lipid Extraction
Beginbycarefullytransferringthedriedsampleintoacleanpulverizationcapsuleequippedwithasteelballbearing.Placethelidontothecapsulebody,makingsurethatitisequippedwithanO-ring.ClampthechamberintotheMixerMill,makingsurethatthereisrubbercushioningoneithersideofthecapsuleinsideoftheclamp.Setthetimerfor3minutesandpushthewhitebuttonlocatedinthemiddleofthetimerdialtopulverizethesample.
WhentheMixerMillhasfinished,removethechamberfromtheMixerMillandremovethelidfromthebodyofthecapsule.Ifthelidhasbecomestucktothebody,lightlytapthecapsuleonitssidesandtryremovingthelidagain.Repeatasmanytimesasittakestoremovethelid.Usingacleanspatula,transferthepulverizedsamplefromthecapsuleandlidbackintothelabeledscintillationvial.Repeatthisprocessforeachsample.
SomesamplesetsmaybelargerthanthenumberofMixerMillcapsuleswecurrentlyhave,sobesuretoproperlycleanthecapsulesbetweenhomogenizationevents.SeeSection7.3.
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7.3 Cleaning the Capsules, Spatulas, and Forceps
Takeallofthedirtycapsules,spatulas,forceps,andpaperclipsintheTo Be CleanedcontainertothesinkinRoom253E.FillthecontainerwithwaterandMicro-90soap.Donotleavethemtosoakovernight,asthepaperclipswillstarttocorrode.
Wearinggloves,removethespatulasandwipethemthoroughlywithasoapygloveorbottlebrush,gettinganyleftovermaterialoffbeforeplacingtheminthesolidstainlesssteelsonicatorinsertfilledwithMicro-90soapandwater.
Removeallofthepaperclipsfromthecontainer,thoroughlyrinsethemwithwater,andplacethemontheabsorbentmaterialbythesinktofullydry.
Removethecapsulebodies,rinsethoroughlywithwater,and,usingasmallscrubbrushcoatedinMicro-90soap,scruboutthebodies.Thoroughlyrinsethebodieswithwaterandplacetheminthesamestainlesssteelsonicatorinsert.
Usingapairofforceps,removealloftheO-ringsfrominsidethecapsulelids,rinsethoroughlywithwater,and,usingasmallscrubbrushcoatedinMicro-90soap,thoroughlyscruboutthelids.Rinsethelidswithwaterandplaceeverythinginthestainlesssteelsonicatorinsert.
ThesteelballbearingsandO-ringsleftatthebottomoftheTo Be Cleanedcontainershouldbethoroughlyrinsedwithwateruntiltheyarefreeofanybiologicalmaterial.Addthemtothestainlesssteelsonicatorinsertandmakesurethatthesoapywatercoverseverythinginthetub.Add1,650mLofwaterintothesonicatorbeforeplacingthesolidstainlesssteelinsertwithallofthepartsandspatulasintothesonicator.Sonicatefor30minutes.Whenthesonicatorisfinished,removetheinsertandrinsewellwithwater,makingsurethatnothingislostdownthesink.Layeverythingoutontheabsorbentpapernexttothesinktodryovernight.Donotleavetheutensilstositovernightinthesoapywaterasthesoapiscorrosive.
Afterallofthetoolshavefullydried,placethembackinthesolidstainlesssteelinsertandfillitwithacetoneinafumehood,makingsureeverythingissubmerged(donotsonicatethepaperclipswiththetools).Makesuretowearappropriategloveswhileworkingwithsolvent.Placethetubbackinthesonicatorafteradding1,650mLofwatertotheunit,andrunforanother30minutes.Whenthesonicatorisfinished,wearinggloves,dumpthesolventwasteintoawastecontainerinthefumehoodanddisposeofitintheappropriateflammablewastejug.Layallofthetoolsoutonacleanpieceoffoilunderthefumehoodtoallowthesolventtofullyevaporate(approximately2hours).Wheneverythingisdry,rebuildthecapsules,placingaballbearingineachone,andplacethecleanspatulasbackwiththerestonthetray.
Afterthepaperclipshavefullydried,placetheminacleanbeakerinthefumehood.Usinganacetonesquirtbottle,triplerinsethepaperclips,drainingthembetweeneachrinse.Letthemsitunderthefumehooduntilallofthesolventhasevaporatedfromthem.Oncetheyhavedried,placethembackwiththerestofthepaperclips.
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8.0 Weighing Samples
Throughoutthisfinalprocess,attentiontodetailiskey.Itisimportanttorememberthattheoilsfromyourhandscancontaminatesamples,soitisbesttoalwaysusecleantoolsandweargloves.Itisalsoimperativetodouble-checksampleweightswheninputtingthemintothetemplate,asanerrorwillthrowoffthecalculationofthesample.
8.1 Building a Loading Sheet
Theloadingtemplatesarefoundonthestableisotopeserver.Selecttheappropriatetemplateandsaveitusingthenameoftheset—forexample,ST1111 Loading.Donotoverwritethetemplate.PlacethenewloadingfileintothesetfolderfoundontheserverunderStable Isotope Projects.Inputthedatafoundinthecorrespondingsetup.csvfileintotherightcorneroftheloadingtemplate(startingwithcellI25).TheinformationwillautofillincolumnZandthroughouttheloadingsheet.Typethesetnameintotheboxatthetopofthepage(cellI3),andfillouttheinformationincellsD52–D56;theinformationwillautofillonthenextpage.
Dependingonhowmanysampleshavebeenassignedtotheset,rowswillhavetoberemoved.Highlighttheemptyrowsthatarenotneededanddeletethem,shiftingthecellsup.Ifthesetissmall,itisnotnecessarytorunallofthestandards.AllsamplesetsmusthavefiveSRMs(tworunatthebeginningoftherun,oneruninthemiddle,andtworunattheendofthesequence),andaminimumof5eachofasparticacidandhistidinestandards(twoeachatthebeginningoftherun,oneeachevery15orfewersamples,andtwoeachattheendofeveryrun).
Oncetheloadingsheetisfullyfilledout,printoffahardcopytofillouttheweightsofyoursamples.TheSample & Standard Weight Guide,foundonthethirdtabintheloadingtemplate,canbereferencedifyouareunsureoftheweightsforthestandardsandsamplesintheset.
8.2 Pilling of Samples for Analysis
Forallpillingoperations,beextremelycarefulnottotouchthetincups,theworkingendsofthetools,orpartsofthemicrobalancewithyourfingers,oryoucouldcontaminatethemwithforeignmaterials.Makesurethatallofthetoolsyouwilluseforpillinghavebeenpreviouslyrinsedwithacetoneandarecleanbeforebeginning.Usingthebalancebrush,makesurethatthebalanceisfreefromanydebris.Labelthelidofa96-wellplatewiththesamplesetnumber.Alwaysbeginfromweighingoutthesamplesofasetbeforemovingontothestandards.Itisbestpracticetoweighthestandardsthedaythatyou
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willrunthemontheIRMS.ThewellplateshouldalwaysbestoredinadesiccatorbetweenpillingsandpriortorunningthesamplesetontheIRMS.
Usingforceps,beginbytakingone4×6tincupfromthecontainerandplaceitonthemicrobalance.Allowthebalancetostabilizewiththedoorclosedbeforetarring.Next,openupthedoorand,usingthesmallsamplespoon,scoopasmallamountofsamplefromthevial,makingsurethatyoudon’tgetanyskin,bones,largechunks,oranythingthatdoesn’tlookhomogenized.Weighouttheappropriateamountofsampleforthematrixyouareworkingwith.Closethebalancedoorandlettheweightstabilizebeforerecordingtheweightofthesampleontheloadingsheet.
Afteropeningthebalancedoorup,useforcepstocrimpthelipofthetincupshutsothatnosampleislostduringfolding.Then,usingtheflat-tippedforceps,foldthetincupintoasmalltightcubeontopofthestonesurface.Makesuretherearenosharppiecesstickingoutofthecubeandthatthetincapsuleisflatsothatthecubedoesnotgetcaughtupontheautosampler.Usingforceps,placethesampleintheappropriatecellinthewellplateandmoveontothenextsample.Makesuretowipedownallofthetoolsalongwiththestonesurfacewithapapertowelbetweeneverysample,soasnottocross-contaminatesamples.Repeatthisprocessforallofthesamplesandstandardsintheset.StorethewellplatesinthedesiccatoruntiltheyarereadytobeloadedontheIRMS.
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Whenyouaredoneusingthetools,thoroughlyrinsetheminacetoneandstorethemawayuntilthenexttimetheyareused.Alwaysplacethelidonthetinthatthecapsulesarestoredinbetweenuses.Thebalanceshouldalwaysbeleftwiththedoorshutbetweenuses.Turnoffthebalanceattheendoftheday.
8.3 Finalizing the Loading Sheet
Beginbyinputtingalloftheweightsofthesamplesandstandardsintotheset’sloadingsheet.Doublecheckthatallweightsweretypedincorrectlybeforemovingon.
Clickonthesecondtabofthetemplate,labeledData,andmakesurethatalloftheinformationfromthefirsttabhastransferredoveraccurately.Ifyouhaveremovedrows,youmighthavetostartatthefirstrowanddragitdownandthenmanuallyremovetherowsthatarenotneeded.Youshouldbeleftwiththesamesequencenumberattheendofthedatatabasonthesetsheet.
SavethisloadingsheettoaUSBsothatyoucanaccessalloftherequiredinformationupstairswhenitistimetoloadthesampleset.Makesurethatyoukeepthehand-writtenloadingsheetintheset’sfolderincasethereisadiscrepancywithsampleweights.
9.0 Loading the Samples on the Elemental Analyzer (EA)
Thedaybeforeyouplanonrunningtheset,checktheinstrumentlogbookformaintenanceneedsandactaccordingly.Ifthesystemrequiresanewreactor,allowthesystemtoequilibrateandflushovernightbeforerunninganyair/watertestsorcheckingthebackgroundoftheinstrument.
9.1 Instrument Maintenance
ThedaybeforeyouplantorunasetontheIRMS,checkthestatusoftheinstrument.Checkhowmanysampleshavegonethroughthewatertrap,reactor,andashtube.Replacethewatertrapafterevery500samples.Replacethereactorafter300samples.Replacetheashtubeafter124samplesand/orbetweensamplesets.
Checkthelevelsonallofthegastanksalongthewall,recordingthemontheclipboardhangingnearthetanks.Ifanytankisbelow500psi,changeitoutwithanewtank,andiftheheliumtankislow,usetheswitchingvalvetoswitchtotheothertankthatshouldbehookedupandopen.Thismaintenanceshouldbedonethedaybeforearuntoallowtheinstrumenttostabilize.YoushouldalsomakesurethatthecorrectinstrumentconfigurationhasbeenselectedinIsodat Acquisitioninthebottom-leftcorner.
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9.2 Running Linearity and On/Offs
Onthemorningofthedayyouareplanningtorunthesampleset,on/offsandlinearitychecksneedtobeperformedforbothcarbonandnitrogen.MakesurethattheConFlo and EAsystemsetuphasbeenselectedinthebottom-leftcornerofIsodat Acquisition.
BeginbyopeningupIsodat Acquisition.SelecttheON_OFFs.seqsequencefromthesequenceslistinthebottom-leftcorner.Everythingisallsettogoonceithasbeenproperlyloaded.ClickthegreenStartbuttonandselecttheOKbuttononthenextscreen.Thesequencewillcontinuethroughalloftheon/offsandlinearitychecks.Ittakesabout2hourstorunthewholesequence.
9.3 Checking the On/Offs and Linearity Checks
Whenthesequencehasfinished,openupIsodat WorkspaceandselecttheResultstab.Openuptheon/offsequencethatwasrun—itwillhavethedateattachedtothefilename.Openeachfilebydouble-clicking.Thestandarddeviations/slopeshouldbecheckedandshouldbelessthanorequalto0.06.Iftheyaregreaterthan0.06,theon/offsshouldberunagain;itmayindicatethatthesystemneedsmaintenance.
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TocalculatetheCO2on/offs,doubleclickonthefiletitledCO2 on_off 4.Selectthelastonetoruninsequence(ittakessometimeforthesystemtostabilize).Whenitopenstothescreen,clickonthecolumntitledd13C/12Ctowardsthebottomofthepage—thisshouldselecttheentirecolumn.Right-clickonthecolumnandselectCalculate.Thestandarddeviationshouldbe≤0.06.
TocalculatetheN2on/offs,double-clickonthefiletitledN2 on_off 4.Thisisthelastonetoruninasequenceofthree.Whenitopenstothescreen,clickonthecolumntitledd15N/14Ntowardsthebottomofthepage—thisshouldselecttheentirecolumn.Right-clickonthecolumnandselectCalculate.Thestandarddeviationshouldbe≤0.06.
TocalculatetheCO2linearity,double-clickonthefiletitledCO2_linearity_(2).MinimizeIsodat WorkspaceandopenLinearity Calculator(anExcelfilelocatedonthedesktop).OpenIsodat WorkspacebackupandselecttheentireAmpl. 44column.CopyandpasteitintotheAmplitude 44columninLinearity Calculator.Next,selecttheentired13C/12CcolumninIsodat Workspace.Copy–pasteitintothed13C/12CcolumninLinearity Calculator.Excelwillcalculatetheslope.Theslopeshouldbe≤0.06.
TocalculatetheN2linearity,doubleclickonthefiletitledN2_linearity_(2).MinimizeIsodat WorkspaceandopenLinearity Calculator(anExcelfilelocatedonthedesktop).OpenIsodat WorkspacebackupandselecttheentireAmpl. 28column.CopyandpasteitintotheAmplitude 28columninLinearity Calculator.Next,selecttheentired15N/14NcolumninIsodat Workspace.Copy–pasteitintothed15N/14NcolumninLinearity Calculator.Excelwillcalculatetheslope.Theslopeshouldbe≤0.06.
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9.4 Checking the Background in the IRMS
Afterthediagnostictestshavebeencalculated,theIRMSbackgroundshouldbechecked.BeginbyopeninguptheBackgroundtemplateonthedesktop.Logthedateandfollowtheinstructionsinthetemplatetorecordallofthenecessarybackgroundmeasurementsintheworksheetforthatdate.Makesurethatyousavewhenyouarefinished.YoushouldalsomakesurethatyouhaveCO2selectedasthereferencegasand44incup2,45incup3,and46incup4(ifyouright-clickoncup2andenter44itwillautofilltherestifCO2hasbeenselectedasthereferencegas).
9.5 Building a Sequence in Isodat
Tobuildasequence,selectFileandthenNewandselectSequence.TypeinthetotalnumberofsamplesandstandardsinthesetandclickOK.Thiswillgenerateablanksequencetemplate.PlugtheUSBcontainingthesamplesetloadingfileintothecomputer.OpenupthefileandclickontheDatatab.BegincopyingtheappropriatedatafromtheDatatabandpastingitintothecolumnsintheblanksequencetemplateinIsodat Acquisition.TheSample NamecolumninformationispastedintotheIdentifier 1columninIsodat Acquisition.TheSample TypecolumninformationispastedintotheIdentifier 2columninIsodat Acquisition.SelecttheentireEA Methodcolumn,right-click,andselectFill grid with Data.SelectthemethodFlash Default\HTN_C_4sec_O2.eam.SelecttheentireMethodcolumn,right-click,andselectFill grid with Data.SelectthemethodMethods\C_N\N_C_karl_jonelle_new.met.
Whenallofthecolumnshavebeenfilledin,makesurethatthePeak Centercolumnhaschecksinalloftheboxesforalloftheruns.Forthefirstblank,selectStart Blank MeanfortheTypecolumn.Forthesecondandthirdblanks,selectAdd Blank MeanfortheTypecolumn.AlloftheothersamplesandstandardsshouldhaveSampleastheType.Right-clickanywhereinthesequenceandselectFit Cells to Grid.
Whenthesequenceiscompletelyfilledout,gouptoFileandselectSave As;namethesequencethenameofthesampleset.Next,gouptoFileandselectPrint.Placethisprintedcopyofthesequenceinthesamplesetfolder.
SelectthegreenStartbuttonandbegintofillouttheset’sinformation.FortheFolder NameselectPreandselectDate.FortheFile NameselectPreandselectIdentifier 1.DeselecttheAutoEnumoption.SelectExcelfortheexportandselectModify Template,add08Dec2016testEAN_CO2.wke,andselectOk.FortheExport File Name,typeinthesetname.Finally,selectInterface Standby After Sequencesothattheinstrumentwillautomaticallygointostandbyafterthelastsamplehasrun.BeforehittingOK,loadtheautosamplerwiththesamplesandmanuallyadvancethetraytodropthefirstpillintothechamber;seeSection9.6.
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9.6 Loading the EA Autosampler
Removeallofthetraysfromthetopoftheautosampler,makingsurethatthegearissettozero(0).Removetheclearlidoffofthefirsttrayandbeginloadingthesamplesintheslotsonebyoneuntilallofthesampleshavebeenloadedinthetrays.Takecarenottoskipaslot,ortoplacetwosamplesinoneslot.Afterallofthesampleshavebeenloaded,stackthetrays,startingwiththe0–31trayatthebottomandsequentiallyaddingallofthetraysontopofeachother.Makesuretoplacetheclearlidontopofthestackoftraysbeforeplacingitontopoftheautosampler.Ensurethatthestackoftraysissittingevenlyandlevelontopoftheautosamplerandmanuallyadvancethefirsttray,turningitclockwiseoneclicktodropthefirstpillintothechamber.Makesurethereisaclickandthatthetrayhasfullyadvancedto1.
9.7 Starting the Run
Afterthesampleshavebeenloadedandthetrayhasbeenadvanced,presstheOKbuttoninIsodat Acquisition(whereyouleftitinSection9.5).Checktomakesurethatyouhaveselectedtoputtheinstrumentinstandbyafterthesetisdonerunning.Alsochecktomakesurethereisn’taflagwavingtoindicatethatthesystemisonlygoingtorunaselectednumberofsamplesoutofthesequence.Itisbesttowatchthefirstsampleruntoensurethateverythingisrunningproperly.Besuretoupdatethewhiteboardbythecomputer,alongwithboththecorrespondingbookandbinder,withthesampleinformationandnumberofsamplesthroughthereactor,watertrap,andashtube.
9.8 Post-Run
Afterthesequenceisfinishedrunning,makesurethatallofthesampleshavedroppedfromtheautosamplerbycheckingthetraysandchamberforanythatmighthavegottencaughtorleftbehind.Resetthetraybymanuallyrotatingthegearbacktozero(0)alongwithallofthetrays.Checkthegastanklevelsandchangeoutanytanksthatmightbelow.Makesurethatthesystemwentintostandbymodeuponcompletionofthesequencebycheckingthattheflowsareturneddowninthemethod.
Ifeverythinglooksgood,usingtheshortcutonthecomputerdesktopcalledEA Results,findtheExcelfilegeneratedforthesetyoujustranandsaveitontheUSByoubroughtupwiththeloadingtemplate.TaketheUSBbackdowntoyourcomputertoprocessusingR.
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10.0 Excess Sample Archival
Allunanalyzedfieldsamplespreparedforbulkstableisotopeanalysiscanbestoredatroomtemperatureindefinitely.SamplesarestoredwithtightcapsinthestableisotopecabinetinRoom253E.Samplesetsshouldstaytogetherinavialflatwiththeappropriatevisiblelabelingforeasylocation.Samplesetinformationshouldbewrittenontheoutsideofeachvialflat.
Sampleswillbearchivedforuptooneyearoraspecifiedtimeperiodafterthedatahavebeenvalidated,asagreeduponbytheprojectleaderandtheprincipalinvestigator(s).
11.0 Processing the Data
StartbyplacingtheExcelfilegeneratedbyIsodatintotheset’sfolderonthestableisotopeserverintheStable Isotope Projectsfolder.Theloadingtemplateandsetupfilesshouldalreadybeinthefolder.MakeacopyofallofthefilesinthetabR Code,andpastethemintoyourfolder.
11.1 Manipulating the Raw Data
BeginbyopeninguptheManipulate raw data.Rfileintheset’sfolder;itdoesnotneedtobesaved.Fillouttheset’snameonline35,replacingtheXXX,andplacethecursoranywhereaboveline35.HitthebuttonRunatthetopuntilawindowopensuptoselecttherawExcelfilethatwasgeneratedbyIsodat;double-clicktoselect.ContinuetohittheRunbuttonuntilitgoesthroughallofthedataandyoureadalinethatsaysSaved.Double-checkthatthereare4peaksforeachrow.Ifthereisanerrormessageortherearelessthan4peaksforeachrow,troubleshootaccordingly.Wheneverythinglooksgood,youcanexitoutoftheprogramwithoutsaving.
11.2 Processing the Data in R
OpenuptheC/N ProcessingfolderandSave Asthenameoftheset.Insertthesetnameonlines8,59,and118.Answerthequestionsonlines61,62,and63.Inserttherundateonline120.Inserttheprojectnameonline128.SavethechangesatthetopofthepageandhitKnit.ReadthroughthePDFfilethatisgeneratedtomakesurethatallQApass.Copy–pastethepast_histidine28_plus_new_run.csv,past_SRM1946_plus_new_run.csv,andpast_aspartic_plus_new_run.csvfilesintotheR Codefolder,replacingtheolderversions.PlacethefolderinJennie’sinboxtobeinputtedintoFileMaker.
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12.0 Data
ThelaboratorywillprovidedatatablesandQAdocumentationsuitableforQAassessment.Alloriginaldataanddatadocumentationdevelopedbythelaboratoryforagivendatapackagewillbekeptbythelaboratoryforatleastoneyearafterthedatahavebeenvalidatedandreported;and,ifrequested,thedatawillbestoredinthecollectionformatforuptofiveyears.
12.1 Data Review
Alldataarereviewedandevaluatedbylaboratorypersonnel.Thisreviewisundertakenbyanalystswhoareresponsibleforensuringthattheanalyticaldataarecorrectandcomplete,theappropriateSOPshavebeenfollowed,andthattheQAresultsmeettheacceptancecriteria.Itistheprojectmanager’sresponsibilitytoensurethatallanalysesperformedbythelaboratoryarecorrect,complete,andmeetprojectDQOs.Theprojectleaderhasfinalreviewauthority.
12.2 Data Storage
ProcesseddatageneratedbyIsodataremaintainedinelectronicfileswithfrequentbackupandstorageontoanexternalharddrive.AllfinalanalyticalresultscalculatedinRaremaintainedinelectronicdatabasefilesonthestableisotopeservermaintainedbyIT,withfrequentfilebackupsandweeklybackupsontotapeforoffsitestorage.BulkstableisotoperesultsarealsostoredintheEnvironmentalChemistryProgram’sFileMakerdatabase.
13.0 Quality Assurance
TheEnvironmentalChemistry’sbulkstableisotopeestablishedqualityassuranceprovidesguidelinesformonitoringanddocumentingthequalityofanalysessothatadesiredlevelofperformancecanbedemonstratedandmaintained.TheseguidelinesarebasedonprotocolsestablishedpreviouslyforspecificprojectsundertheNationalOceanicandAtmosphericAdministration(NOAA)andtheEnvironmentalProtectionAgency(EPA),andhavebeenadaptedtonewtypesofanalysesandcurrenttechnologies(Sloanetal.2019).
13.1 Quantitation Range
FortheEA/IRMSmethod,theδ13Candδ15NvaluescanbeaffectedifthemassspectrometerresponsesfortheCO2andN2peaksaretoosmallortoolarge.Forfieldsamples,resultsarereportedifpeakamplitudesforN2(mass28and29)andCO2(mass44and46)arebetween500and12,000mV.Samplesthatdonotmeettheabovecriteriaarereanalyzed.Ifpeakamplitudesareneartheirlimits,theaccuracyoftheresultislesscertain.SampleresultsarefootnotedtobeusedwithcautionifthepeakamplitudesforN2(mass28or29)orCO2(mass44or46)arebetween9,000and12,000mVorbetween500and750mV.Thecorrespondingdeltaandweightpercentage(Wt%)measurementswillnotbereportedifthesample’speakamplitudeformass28ormass29is>12,000mVorifitspeakamplitudeformass44ormass46is<500mV.
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13.2 Instrument Calibration
TheEA/IRMSmethodrequiresatleasttwodeltalevelsofcalibrationstandards(histidineandasparticacid,preparedin-house)foranalyteratiomeasurements.Theδ15Nandδ13CvaluesforthecalibrationstandardsareassignedusingprimarystandardmaterialsfromIAEA(IAEACH-7)andtheUnitedStatesGeologicalSurvey(USGS40andUSGS41a).δ15Nandδ13Cmustbecalculatedusinglinearcalibrationandatleastfivereplicateanalysesofeachcalibrationstandard,includingatleastoneofeachatthebeginningandendofthebatchafterextremepoints(ifany)areidentifiedandexcludedduringthecontinuingcalibrationverification(Section13.3).Theδ15Nresultsandδ13Cresultsfortheincludedreplicateanalysesofthecalibrationstandardsversustherespectiveassignedδvaluesmusthaveacorrelationofr>0.9900;ifthiscriterionisnotmet,thenthesamplesinthebatchmustbereanalyzed.
MinimumFrequency:Atleasttwoofeachcalibrationstandardatthebeginningandendofeverybatch,andbetweenevery15orfewerfieldsamples.
13.3 ContinuingCalibrationVerification(CCV)
FortheEA/IRMSmethod,theCCVstandards’δ15Nandδ13Cvaluesareevaluatedusingtheapplicablefourstepsasfollows:
1. TheamplitudesofN2(mass28and29)peaksmustbebetween500and12,000mVandtheamplitudesofCO2(mass44and46)peaksmustbebetween500and12,000mV;otherwise,thatanalysisofthestandardisexcludedfromthedatasetandnotfurtherevaluated.Atleastoneanalysisofeachstandardmustremainatthebeginningandendofthebatch.
2. Thestandarddeviationofδvaluesinthereplicateanalysesofeachstandardmustbe≤0.25permil(‰)forδ15Nand≤0.35‰forδ13C;otherwise,extremepointswillbeidentifiedandremoved(seeStep3).
3. AnextremepointisdefinedastheCCVstandardwiththegreatestdifferenceinδ15Nandδ13CfromthemedianofallreplicateCCVstandards.ExtremepointsareidentifiedandexcludedinastepwiseprocessuntilthestandarddeviationsmeetthecriteriainStep2.
4. Nomorethan20%ofδ15Norδ13CvaluesinthereplicatesofeachCCVstandardcanbeexcludedduetoextremevalues.Atleastoneanalysisofeachstandardmustremainatthebeginningandendofthebatch.
5. MinimumFrequency:Atleasttwoofeachcalibrationstandardatthebeginningandendofeverybatchandbetweenevery15orfewerfieldsamples(samestandardsasthoseusedforinstrumentcalibration).
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13.4 Reference Materials
ThereisnotissueSRMcertifiedforratiosofstableisotopesofcarbonandnitrogen.However,NationalInstituteofStandardsandTechnology(NIST)SRM1946(fishmuscletissue)isusedasanIRM.Theassignedreferencevaluesforδ15N,δ13C,Wt%N,andWt%CforSRM1946arethemeanofrepeatedin-houseanalysesofthisIRMforstableisotopesofcarbonandnitrogen.Thelaboratory’sperformancefortheEA/IRMSmethodisconsideredacceptableifaminimumofthreesamplesofIRMperbatchmeetfourcriteriaasfollows:
1. ForeachIRMsample,theamplitudesofN2(mass28and29)andCO2(mass44and46)peaksmustbebetween500and12,000mV;otherwise,thatsampleoftheIRMisexcludedfromthedatasetandnotfurtherevaluated.
2. Themeanoftheδ15Nvaluesandthemeanoftheδ13Cvaluesmustbewithintheirrespectivecontrollimits(Equations7–10).
3. Thestandarddeviationoftheδ15Nvaluesmustbe≤0.3‰andthestandarddeviationoftheδ13Cvaluesmustbe≤0.2‰.
4. ThemeanoftheWt%NvaluesandthemeanoftheWt%Cvaluesmustbewithintheircontrollimits(Equations11and12).
Ifbothδ15Nandδ13CmeettheacceptancecriteriabutaWt%doesnot,thentheδ15Nandδ13CarereportedforallsampletypesinthebatchbutWt%andC/Nratioarenot.
Equations:
• δ15Nuppercontrollimit=referencevalue+0.3‰ (7)• δ15Nlowercontrollimit=referencevalue−0.3‰ (8)• δ13Cuppercontrollimit=referencevalue+0.2‰ (9)• δ13Clowercontrollimit=referencevalue−0.2‰ (10)• Wt%uppercontrollimit=(1.05×Wt%referencevalue) (11)• Wt%lowercontrollimit=(0.95×Wt%referencevalue) (12)
MinimumFrequency:Tworunatthebeginningandendofeverybatchandoneruninthemiddle,withaminimumofthreesamplesofIRMperbatchmeetingtheacceptancecriteria.
13.5 Method Blanks
FortheEA/IRMSanalyses,themethodblanksaretincupswithnosampleadded,whichareanalyzedinthesamemannerastheenvironmentalsamples.Theseblanksareusedtocorrecttheδ15Nandδ13Csamplevaluesfortracesofnitrogenorcarbonmaterialsinthetincups,andarenotameasureofcontaminationthatoccurredduringsampleprocessing.TheN2mass28andCO2mass44peakamplitudesforallofthemethodblanksmustbe<50mV,orthesourceofcontaminationmustbedeterminedandcorrectiveactiontaken.
MinimumFrequency:Threeatthebeginningofeverybatch.
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13.6 Sample Replicates
ForEA/IRMSanalyses,replicatesamplesoftheIRMareanalyzedbetweenevery15orfewerfieldsamples,withaminimumofthreeperbatch,toshowtheperformanceoftheEA/IRMSsystem.Duplicateortriplicatefieldsamplesaresuggestedforapproximatelyevery10fieldsamples,butarenotusedforQA.Therearenoacceptancecriteriaforreplicatefieldsamplesbecausemanyexplanationsexistforwidelyvaryingvalues(e.g.,problemswiththesampleprocessingorthehomogeneityofthestartingsample)thatareoftenoutsidethecontroloftheanalyticallaboratory.However,within-samplevariabilityoftheresultsforreplicatefieldsamplesmaybeusefultotheresearcher.
13.7 Reported Results
Forstableisotoperatios,δ15Nandδ13Cvaluesarereportedas‰,whereδ13isthedifferenceinratioofcarbonisotope13Ctocarbonisotope12CinasamplerelativetothatinthePeeDeeBelemnitestandard,andδ15Nisthedifferenceinratioofnitrogenisotope15Ntonitrogenisotope14Ninasamplerelativetoatmosphericnitrogenusedasthestandard(Equations13and14).
Equations:• δ13C={[(13Csample/12Csample)/(13Cstandard/12Cstandard)]–1} (13)• δ15N={[(15Nsample/14Nsample)/(15Nstandard/14Nstandard)]–1} (14)
Forstableisotoperatios,Wt%N,Wt%C,andC/Nratiosarealsoreported.
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ReferencesHerman,D.P.,D.G.Burrows,P.R.Wade,J.W.Durban,C.O.Matkin,R.G.LeDuc,L.G.Barrett-Lennard,
andM.M.Krahn.2005.FeedingecologyofeasternNorthPacifickillerwhalesOrcinus orcafromfattyacid,stableisotope,andorganochlorineanalysesofblubberbiopsies.MarineEcologyProgressSeries302:275–291.
Sloan,C.A.,B.Anulacion,K.A.Baugh,J.L.Bolton,D.Boyd,P.M.Chittaro,D.A.M.daSilva,J.B.Gates,B.L.Sanderson,K.Veggerby,andG.M.Ylitalo.2019.QualityAssurancePlanforAnalysesofEnvironmentalSamplesforPolycyclicAromaticHydrocarbons,PersistentOrganicPollutants,DioctylSulfosuccinate,EstrogenicCompounds,Steroids,HydroxylatedPolycyclicAromaticHydrocarbons,StableIsotopeRatios,andLipidClasses.U.S.DepartmentofCommerce,NOAATechnicalMemorandumNMFS-NWFSC-147.https://doi.org/10.25923/kf28-n618
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Appendix A: Instructions for Submission of Samples
for Stable Isotopes Analyses
NWFSC Environmental Chemistry Program
1. Priortofillingoutanypaperwork,pleasediscussyourprojectwithPaulChittaro,(206)861-7617,and/orJonelleGates,(206)302-2445.
2. Foranideaofsampleweightsrequiredforstableisotopeanalysis,refertothistable:
TypeMinimum Sample Weight for Bulk C/N
Minimum Sample Weight for CSIA
WholeBodyFish 1g 3gFishMuscle 1g 3gMussel/Shellfish 2g 3gMarineMammalskin/Blubber 0.5g 1gVegetation 2g 3gInvertebrates 1g 3g
3. ObtaintheSubmissionFormforStableIsotopesAnalyses1(see Appendix B)fromthelinkprovided,orviae-mailfromJonelleGates([email protected])orJennieBolton([email protected]).Thisinformationwillbeusedtoscheduletheanalyses,trackthedataandresults,andanticipateproblems.Note:Non-NOAApersonnelmayneedtorequestpermissiontoaccesstheform.
1https://drive.google.com/file/d/1Zan5tFQUOtoMvsyRkdfhtAcpkV2SRNUl/view
4. Filloutinformationaboutyourprojectandthegeneralnatureofyoursamplesonthefirstpageoftheform(theProject Informationtab).• Investigator:ThepersonatNWFSCsubmittingthesamplesforanalysis,orthe
contactpersonforanexternalagency.• Project Name:Aconcise(4–8word)namefortheproject,includingthe
samplingyear,especiallyiftheprojectismulti-year.• Project Description:Abriefdescriptionofwhattheprojectisabout(nomore
thanaparagraphisneeded).• Charge Code:Thebillingcode,contractnumber,etc.,fortheworktobedone.• Referring Agency:EithertheProgram/DivisionatNWFSC,orthenameofthe
externalagency/office.• Date of Request:Dateonwhichtherequestforanalysisismade,afterthesamples
areallavailabletoNWFSCpersonnelandanyfinancialagreementsarefinalized.• Date Data are Needed:Thisdateshouldbesetbyagreementoftheinvestigator
andNWFSCpersonnel,dependingonprioritiesandworkload.Ifyouhaveameetingwheredatawillbepresented,pleaseincludethisinformationtohelpsetthelaboratorypriorities.Wewilltrytoaccommodatetheserequestsasbestwecan.
• Analyses Requested:Checktheboxesforeachmeasurementrequired.
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• Sample Prep. Required:Checktheseboxesforanysamplepreparationthatwillnotbedonebyyourgroup.Normally,thegroupsubmittingsampleswillfreeze-dryandpulverizesolidsamplesaccordingtoourrecommendations(NOAAProcessedReportNMFS-NWFSC-PR-2020-04,Standard Operating Procedures for Measuring Bulk Stable Isotope Values of Nitrogen and Carbon in Marine Biota by Isotope Ratio Mass Spectrometry (IRMS),willbeprovided),andweighthesamplepillsalongwithanappropriatenumberofstandardpills.AdditionalchargeswillbeincurredtotheinvestigatorifNWFSCpersonnelarerequiredtodothisprepwork.Weadvisegroupssubmittingsamplestofreeze-dryandpulverizesolidsamplesaccordingtotheprovidedSOP.Ifyourgrouphasnoexperienceinpillingsamplecapsulesforstableisotopeanalysis,EnvironmentalChemistrypersonnelcanweighthefinalsampleandpreparethecapsulesforanalysis(subjecttoagreement).
• General Description and Number of Samples:Providesampledescription(matrix)andnumberofsamplestobeanalyzed.
• What Precision is Required (in per mil) for the Analyses:Ifknown,pleaseindicatetheprecisionfortheanalysis.Atrophiclevelrepresentsadifferenceof~3‰ford15N.Differencesdownto0.5‰canbemeasured,butthisrequiresalargernumberofreplicatestobeanalyzed.
• Expected %N Range:Ifknown,pleaseprovidetherangeofvaluesexpected.Sampleswithveryhighorverylowvaluesmayneedtobepreparedoranalyzeddifferentlyfromothersamples,sothisinformationneedstobeknowninadvanceanddiscussedwithNWFSCpersonnel.
• Expected %C Range–ifknown,pleaseprovidetherangeofvaluesexpected.Sampleswithveryhighorverylowvaluesmayneedtobepreparedoranalyzeddifferentlyfromothersamples,sothisinformationneedstobeknowninadvanceanddiscussedwithNWFSCpersonnel.
• Anything Unusual About Samples:Listanypropertiesofanyofthesamplesthatmightbeexpectedtocauseproblemswiththeanalyses.Forexample,homogeneitybecomesaproblemwithsamplesthathaveahighpercentageoflipid.Weadviselipidextractionortheuseofliquidnitrogeninthesecasesbeforehomogenization.Anysamplesexpectedtobelessthan5%nitrogenshouldbenoted.ProblemsamplesshouldbediscussedwithPaulChittaroorJonelleGatesattheoutset(priortosamplepreparation).
• In What Format Would You Like the Final Data: Checkwhetheryouwouldlikehardcopieswithtables,anExcelworksheet,orboth.
5. FilloutinformationaboutyoursamplesontheSample Informationtabofthetemplate.Notethatofthefollowinginformation,thosemarked*areabsolutelyrequired;theotherswillappearonthedatareportyoureceiveandwillaidusintrackingandidentifyingyoursamples,andinprovidingtheresultsinamorecompleteformat.Onehundredsamplerowsaregiveninthistab,butyoumaysubmitmoresamplesifyouhavethem.Makethesheetaslongasyouneed.• Sample Category:*Selectoneofthefollowing(pleasedon’tabbreviate):
vegetation,fish,invertebrate,marine mammal,filtrate.• Sample Type:*Describeeachsampleasfollows:Tissue – skin,Tissue – muscle,
Tissue – whole body,Tissue – fat,Tissue – egg,Tissue – blubber,wood,leaf,berry,bark,periphyton,algae.Forsamplesfromtheanimalkingdom,pleaseusetheformatTissue (space) (dash) (space) typeasshownintheexamplesabove.ContactJennieifyouhaveanyquestions.
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• Site:Wherethesampleisfrom.Examples:Snohomish Station 3,Prince William Sound.Ifknown,pleaseincludethelatitudeandlongitudecoordinatesofwherethesampleswerecollected.
• Species:PleaseprovidetheLatingenusandspeciesnamesifpossible,ortolowestlevelofclassification(genus,family,order).Examples:Tsuga heterophylla,Thuja plicata,Oncorhynchus tshawytscha,Tricoptera.
• Common Name:Thecommonnameofthespecies.Examples(fromLatinnamesabove):western hemlock,western red cedar,Chinook salmon,caddis fly.
• Field/Animal ID:*Yourfieldidentifier/specimennumberforthissample.Examples:SN3-0095-02,006,145-SNOmmmp.
• Jar/Vial #:Ifthesamplecontainerhassomeadditionalidentifyinginformation,enterthatinformationhere.Thisisatextfield,soanyshortcombinationoflettersandnumbers(excludingspecialcharacters)isallowed.Youcouldalsousethisfieldtodesignateatreatmentgroup.Examples:1 of 2,4cc WX.
• Life Stage:Examples:adult,larvae,fry,juvenile,lactating,pregnant,spawning.• Collection Date:Datethesampleswerecollectedinthefield,ifyouwouldlike
thisinformationtobedisplayedonyourreportofresults.• Sample Weight:*Weightofthesamplepilledforanalysis,ifyouarepillingyour
owncapsules.• Notes:Noteanythingaboutthesamplesthatmaycauseproblemswiththe
analyses(forexample,individualsampleswithnitrogencontentlessthan5%couldbenotedaslow N).
• Sample Location/Contact:Letusknowwhomtocontactwhenwearereadytoanalyzeyoursamples,sotheycanshowuswherethesamplesarelocated.
• Cassette Identification:*Identifyeachcassetteincludedinyoursubmission.Aresearcher,group,and/orprojectnameisagoodidea,alongwithanumber.Example:Elwha River Nutrients 2010.
• Cassette position:*Providethewellnumber(A1,C3,etc.)foreachsampleinitscassette.Putthepositioninasinglespreadsheetcolumn(e.g.,don’trecordthepositionwithanAinonecolumnanda5inanother;recordthepositionasA5).Youmayaddcolumnsasneeded;forexample,forReachorTransect,andwewillaccommodatetheseinreportingtotheextentpossible.Important:Aftercompletingthelistofsamples,checkoverthelisttoensurethat:1) Emptypillsorpillsthathavetoolittlesampletoeffectivelyanalyze(check
withJonelleorPaulifyouhavequestions)areremovedfromthelist.2) ThesamplelistissortedfirstbyCassette IDandthenbyCassette position.3) Therearenospecialcharacters(i.e.,/,*,?,‘and,).Youcanuseanunderscore
(_)toseparatedescriptions,ifneeded.Specialcharactersaredifficulttocheckforwhenloadingsamples,andtheywillcausearuntimeerrorintheinstrumentduetoOSlimitations.
WerecommendthatsamplesbesubmittedinordersothattheloadingoftheSIautosamplergoessmoothly.Thiswillgreatlyreducethechanceoferrorsfromsamplesbeingloadedinthewrongorder,assamplesareusuallyloadedexactlyintheorderpresented.Theautosamplercanhold124samples/standardsinonerun.Thesubmittedlistofsamplesmaybebrokenupintomultiplesetsrunoverseveraldays;youwillneedtoensurethatsufficientstandardandSRMpillsarealsosubmitted.
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6. Ifyouhaveadditionalinformationthatyouwouldliketohavedisplayedaspartofyourreportofthestableisotoperesults,orifyouneedthedatapresentedorprovidedinaparticularformat,pleasediscussyourneedswiththedatamanager,JennieBolton([email protected],(206)860-3359,Room248E).
7. OncetheExcelsubmissionformhasbeenfilledout,emailacopytoJennieBolton([email protected])sothatthesamplescanbescheduledforanalysis,accordingtotheprioritiessetinStep4.
8. Asequencewillbegeneratedbasedontheinformationprovidedalongwithsomebasicpillinginstructionsforthesample/standardpreparer,printedoff,andhandedtothepreparerpriortopilling.
9. IfthesamplesarefromamaterialthathasnotbeenanalyzedbeforebytheEnvironmentalChemistryProgram,youmaybeaskedtoprepareseveraltestsamplessothatanoptimalsampleweightrangeforthematerialcanbeestablished.
10. If collecting your samples requires filtration:• Makesuretoonlyuseglass-fiberfilters.Do not use paper filters.• Trytogetaslittleoftheglass-fiberfilterintothesampleaspossible.Piecesof
glassfibercancauseproblemswiththeinstrumentduringtheanalysis.• Saltfromseawatercancompromisetheanalysis.Whencollectingsamplesin
seawater,trytogetaslittleofthewateraspossibleinwiththesamplebeforefreeze-drying.
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Appendix B: Submission Forms for Stable Isotopes Analyses
Project InformationProject Information
Fill out the following information regarding your request. See the Instructions for more information.
Investigator:
Project Name:
Project Description:Charge Code:
Referring Agency:Date of Request:
Date Data are Needed †: Data Manager use only
Analyses Requested: %C %N C/N Ratio ∂13C ∂15N Date in projects db:
Extract Lipid Freeze Dry PulverizeSample Prep Required: Date in whitesheet db:
General Description and Number of Samples: Edit: 9/13/2004 JLBWhat Precision is Required (in per
mil) for the Analyses?*Expected %N Range:Expected %C Range:
Is %N Content Less Than 5% for Any Samples?
Anything Unusual About the Samples:
In What Format Would You Like the Hard copy data tables Excel worksheet BothFinal Data?
* A trophic level is a difference of ~ 3 per mil, but measurements down to 0.5 per mil can be made using a suitable numberof replicate analyses.† "ASAP" is not a date - please see the submission instructions.
Submission Form for Stable Isotopes Analyses
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Sample Information
* Indicates information that is required - please see submission instructions for how to format the entries.
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Sample Information
* * * * * *Sample Category Sample Type Site (include Lat./Long. if possible) Species Common Name Field/Animal ID Cassette ID Vial/Well # Life Stage Collection Date Sample Wt (mg) Notes Sample Location/Contact
123456789
1011121314151617181920212223242526272829303132333435363738394041424344454647484950
525354
Submission Form for Stable Isotopes Analyses
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U.S. Secretary of Commerce
Wilbur L. Ross, Jr.
Acting Under Secretary of Commerce for Oceans and Atmosphere
Dr. Neil Jacobs
Assistant Administrator for Fisheries
Chris Oliver
February 2020
fisheries.noaa.gov
OFFICIAL BUSINESS
National Marine Fisheries ServiceNorthwest Fisheries Science Center2725 Montlake Boulevard East Seattle, Washington 98112