CalibrationCalibration ofof MechanicalMechanical PipettesPipettes inin thethe ModernModern WastewaterWastewater LaboratoryLaboratory DeWayneDeWayne KennedyKennedy--ParkerParker andand RogerRoger SchultzSchultz 1 Copyright 2007, University of Wisconsin Board of Regents AnyAny referencereference toto productproduct oror companycompany namesnames doesdoes notnot constituteconstitute endorsementendorsement byby thethe WisconsinWisconsin StateState LaboratoryLaboratory ofof Hygiene,Hygiene, oror thethe UniversityUniversity ofof Wisconsin.Wisconsin. DisclaimerDisclaimer 2 Class A volumetric pipettes • Are very accurate and precise • Do not require periodic calibration • Have defined volumes • Are usually broken if dropped • Require practice to master the technique of use with a bulb 3 But, maybe it’s time to go “Mechanical”. OneOne adjustableadjustable volumevolume mechanicalmechanical pipettepipette cancan replacereplace These are fine. aa numbernumber ofof singlesingle volumevolume pipettes.pipettes. 4 MechanicalMechanical airair--displacementdisplacement pipettespipettes areare becomingbecoming moremore commonlycommonly usedused inin thethe modernmodern wastewaterwastewater laboratorylaboratory duedue toto theirtheir manymany advantagesadvantages overover traditionaltraditional glassglass pipettes.pipettes. 5 6 5 - 27 WhereWhere wouldwould mechanicalmechanical pipettespipettes bebe usedused inin aa wastewaterwastewater laboratory?laboratory? • ChlorineChlorine TestingTesting • PhosphorousPhosphorous Testing,Testing, essentialessential ifif usingusing TestTest--nn--tube®tube® methodmethod • StandardsStandards PreparationPreparation • AnyAny pipettingpipetting operationoperation upup toto 1010 mLmL thatthat isis repeatedrepeated oftenoften inin thethe laboratorylaboratory 7 MechanicalMechanical airair displacementdisplacement pipettepipette operation.operation. OperatingOperating buttonbutton isis pressedpressed toto thethe firstfirst stop,stop, movingmoving thethe pistonpiston andand expellingexpelling aa specifiedspecified volumevolume ofof airair fromfrom thethe tiptip 8 After immersing the tip into the liquid, the operating button is released. This creates a partial vacuum and the specified volume of liquid is aspirated into the tip. 9 When the operating button is pressed to the first stop again, the air dispenses the liquid. To empty the tip completely, the operating button is pressed to the second stop (blow out). 10 CostsCosts areare higher,higher, initiallyinitially • MostMost classclass AA glassglass volumetricvolumetric pipettespipettes $10$10 toto $40$40 (( dependingdepending onon thethe size)size) • MechanicalMechanical (non(non--electronic)electronic) $200$200 toto $400$400 • ElectronicElectronic (( multimulti--functionfunction )) $400$400 toto $600$600 11 AdvantagesAdvantages • HighlyHighly preciseprecise andand accurateaccurate • AdjustableAdjustable volumes.volumes. • DisposableDisposable tips.tips. NoNo needneed forfor washing,washing, savingsaving analystanalyst time.time. EliminatesEliminates possibilitypossibility ofof carryovercarryover betweenbetween samples.samples. • PossiblePossible laboratorylaboratory savings.savings. DespiteDespite higherhigher initialinitial investment,investment, costcost maymay bebe lowerlower overover timetime duedue toto savedsaved analystanalyst timetime andand lowerlower replacementreplacement raterate thanthan easilyeasily brokenbroken glassglass pipettes.pipettes. 12 DisadvantagesDisadvantages • HigherHigher initialinitial costcost • FailureFailure toto maintainmaintain andand calibratecalibrate maymay leadlead toto grossgross errorserrors andand poorpoor testtest resultsresults beingbeing reportedreported onon dischargedischarge monitoringmonitoring reports.reports. • FailuresFailures maymay bebe “unpredictable“unpredictable inin timingtiming andand undetectableundetectable byby thethe user.”user.” PeriodicPeriodic calibrationcalibration andand preventivepreventive maintenancemaintenance are,are, therefore,therefore, essentialessential toto ensureensure thethe integrityintegrity ofof laboratorylaboratory results.results. *George Rodrigues, PhD, ARTEL 13 CalibrationCalibration ApproachesApproaches •• GravimetricGravimetric –– mostmost commoncommon • AllAll youyou needneed isis aa calibratedcalibrated analyticalanalytical balancebalance capablecapable ofof measuringmeasuring downdown toto 0.10.1 mgmg (same(same asas forfor totaltotal suspendedsuspended solids)solids) • ThermometerThermometer • RoomRoom temperaturetemperature distilleddistilled waterwater • AA lightlight weighingweighing vesselvessel forfor weighingweighing samplesample replicatesreplicates • Clean,Clean, unusedunused pipettepipette tipstips • SpreadsheetSpreadsheet oror printedprinted formform forfor recordingrecording andand calculatingcalculating pipettepipette precisionprecision andand accuracyaccuracy 14 CalibrationCalibration ApproachesApproaches •• PhotometricPhotometric –– notnot asas commoncommon • RequiresRequires anan expensiveexpensive instrumentinstrument ($5,000)($5,000) • ExpensiveExpensive reagentsreagents 15 Annual Pipette Calibration (gravimetric) Date Analyst Balance Temp. (C) Z-Factor #N/A Serial # Pipette # 117205 Pipette # Pipette # VOLUME WEIGHT VOLUME WEIGHT VOLUME WEIGHT (ml) (g) (ml) (g) (ml) (g) MEAN #DIV/0! MEAN #DIV/0! MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! % CV #DIV/0! % CV #DIV/0! % CV #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! Pipette # Pipette # Pipette # VOLUME WEIGHT VOLUME WEIGHT VOLUME WEIGHT (ml) (g) (ml) (g) (ml) (g) MEAN #DIV/0! MEAN #DIV/0! MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! % CV #DIV/0! % CV #DIV/0! % CV #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! Corrective Action Due To Failures? Acceptance Criteria Spreadsheet created by: % Inaccuracy must be less than 2.00 Donna Johnsen, WSLH No replicate may be greater than 2% from true volume. %CV must be less than 1.00 16 Quarterly Pipette Calibration (gravimetric) Date Analyst Balance Ae 240 Temp. (C) Z-Factor #N/A Serial # J52053 Pipette # Pipette # Pipette # VOLUME WEIGHT VOLUME WEIGHT VOLUME WEIGHT (ml) (g) (ml) (g) (ml) (g) MEAN #DIV/0! MEAN #DIV/0! MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! % CV #DIV/0! % CV #DIV/0! % CV #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! Pipette # Pipette # Pipette # VOLUME WEIGHT VOLUME WEIGHT VOLUME WEIGHT (ml) (g) (ml) (g) (ml) (g) MEAN #DIV/0! MEAN #DIV/0! MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! CORR. MEAN #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! STD DEV #DIV/0! % CV #DIV/0! % CV #DIV/0! % CV #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! % INACC. #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! PASS / FAIL ? #DIV/0! Corrective Action Due To Failures? Acceptance Criteria Spreadsheet created by: % Inaccuracy must be less than 2.00 Donna Johnsen, WSLH No replicate may be greater than 2% from true volume. %CV must be less than 1.00 17 Temperature Z-Factor 15 1.002 15.5 1.002 16 1.0021 16.5 1.0022 17 1.0023 17.5 1.0024 18 1.0025 18.5 1.0026 19 1.0027 19.5 1.0028 20 1.0029 20.5 1.003 21 1.0031 21.5 1.0032 22 1.0033 22.5 1.0034 23 1.0035 23.5 1.0036 24 1.0037 24.5 1.0038 25 1.0039 18 Date 10/25/07 Analyst DKP Balance Ae 240 Temp. (C) 22 Z-Factor 1.0033 Serial # J52053 19 Pipette # 117205 VOLUME WEIGHT (ml) (g) 2.50 MEAN #DIV/0! CORR. MEAN #DIV/0! STD DEV #DIV/0! % CV #DIV/0! % INACC. #DIV/0! PASS / FAIL ? #DIV/0! 20 21 22 23 24 25 Pipette # 117205 VOLUME WEIGHT (ml) (g) 2.50 2.511 2.508 2.514 2.517 2.525 2.514 2.521 2.503 2.496 2.517 MEAN 2.51257 CORR. MEAN 2.52086 STD DEV 0.00858 % CV 0.340383825 % INACC. 0.83445924 PASS / FAIL ? PASS 26 MEAN 2.51257 CORR. MEAN 2.52086 STD DEV 0.00858 % CV 0.340383825 % INACC. 0.83445924 PASS / FAIL ? PASS Acceptance Criteria % Inaccuracy must be less than 2.00 No replicate may be greater than 2% from true volume. %CV must be less than 1.00 27 CalculationsCalculations Mean Weight = sample replicate 1 + sample replicate 2 + (etc.) Number of replicates Mean Volume (Corrected Mean) = mean weight × Z-Factor % Inaccuracy = [(Corrected Mean – true value) ÷ true value] × 100 % CV (Coefficient of Variation) = (Standard Deviation ÷ Corrected Mean) × 100 28 Volume Inaccuracy Imprecision 1 µl ±2.5% ≤ 1.8% 2 µl ±2.0% ≤ 1.2% 5 µl ±1.5% ≤ 0.8% 10 µl - 15 µl ±1.0% ≤ 0.5% 20 µl - 40 µl ±0.8% ≤ 0.3% 50 µl - 90 µl ±0.7% ≤ 0.3% 100 µl - 200 µl ±0.6% ≤ 0.2% Temperature: 20° C - 25° C, constant to ± 0.5° C # of measurements: 10 29 Courtesy of Eppendorf AA pipettepipette isis onlyonly asas goodgood asas it’sit’s operator.operator. 30 Courtesy: ARTEL AA littlelittle trainingtraining cancan gogo aa longlong way.way. 31 Courtesy: ARTEL 32 Courtesy: ARTEL PipettePipette MaintenanceMaintenance •• MechanicalMechanical pipettespipettes mustmust bebe inspected,inspected, cleanedcleaned andand maintainedmaintained regularlyregularly toto assureassure properproper operation.operation. •• ReferRefer toto thethe pipette’spipette’s instructioninstruction manualmanual forfor guidanceguidance onon cleaningcleaning andand maintenance.maintenance. 33 • CareCare mustmust bebe takentaken toto notnot aspirateaspirate anyany