How To Calibrate Your Micropipette

In Steps, How to check and calibrate micropipette

1. How
To
Calibrate
Your
Micropipette
?

2. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Principle
•Micropipettesareprecisionlaboratoryinstruments.
•Theyrequireroutinemaintenanceandcalibration
toensureaccuracyandprecision.
•Usinggravimetricmethodologythisisdetermined
byusingananalyticalbalanceandweighingthe
amountofwaterdeliveredbythepipette.Forthe
purposeofcalibration,milligramsandmicroliters
areconsideredtobeequivalent.
•Calculationofthestandarddeviationandthe
coefficientofvariationareusedtodeterminethe
accuracyandprecisionofeachpipette.
Dr. TamerSoliman

3. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Dr. TamerSoliman
Frequency
•Before pipettes are newly placed into service
•Periodically; suggested interval is quarterly or
according to manufacturer guidelines
•After cleaning
•Anytime a pipette becomes dismantledand
repaired
•As a means of troubleshootingassay problems

4. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Requirements
•Micropipettesfor calibration
•Analytical balance with maintenance completed
•Pipette tips for appropriate pipette volumes
•Weigh boats
•Worksheetfor Micropipette Calibration
•Room temperature water in a clean glass beaker
(Some labs use Peanut Oil)
•Thermometer
•Calculatorwith statistical program, computer with
statistical software, or standard calculator.
Dr. TamerSoliman

5. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calibration
Thecompletecalibrationconsistsoftestingboth
accuracyandprecisionofallmicropipettesoutlinedin
thisprocedure.
GuidelinesforAcceptableCalibrationResults:
•Precisionisbasedontheguidelinessuggestedby
themanufacturerinthepipettepackageinsert.
•Acceptableaccuracyrangesforpipettesshouldbe
basedonthemanufacturer’srecommendations.
•Accuracyrangesofagoodguide:
plusorminus4%forpipettes5–50µl
plusorminus2%forpipettes51–1000µl
Dr. TamerSoliman

6. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calibration
Dr. TamerSoliman
Variable Micropipette
20–200 µl
20µl
= Low-volumesetting
200 µl
= High-volume setting
100µl
= Mid-volumesetting
Forvariablemicropipettes,
calibrationshouldbedoneat3differentlevels
Example

7. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
ProcedureFlowchart
Dr. TamerSoliman
UsingGoodPipetting Technique
Un-Calibrated
MicroPipette
Mid-VolumeSetting
MeasureRe-measure High-VolumeSetting
Low-VolumeSetting
Adjust
Atlow-VolumeSetting
UsingPipetteCalibrationTool
Re-calibrate
IfNotOK
Calculate
both
Precision
and
Accuracy
Perform
Routine Maintenance
Re-calibrate
IfNotOK
MeasureRe-measure
MeasureRe-measure
Calibration
Remove the pipettefrom service
IfNotOK

8. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
Dr. TamerSoliman
ForPipettes5–100µl
1.Recordambientroomtemperaturetodetermine
waterdensitytobeusedincalculations.
2.Placeaplasticweighboatandpipettetiponthe
balanceandtarethebalance(adjusttoZero).
3.Placethetipontothepipetterequiringcalibration.
PrewetthenPipetteroomtemperaturereagentgrade
waterintothetipusinggoodpipettingtechnique.
4.Turnthepipetteintoahorizontalpositionandremove
thetiptakingcarenottoloseanyofthewater.
5.Weighthetipandwateronthesameplasticweigh
boatandrecordtheresult.
6.Repeatsteps10–20times(dependingonthecriteria
forprecisionestablishedbyyourlaboratory).

9. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
Dr. TamerSoliman
ForPipettes100–1000µl
1.Recordambientroomtemperaturetodetermine
waterdensitytobeusedincalculations.
2.Placeaplasticweighboatontoananalyticalbalance
andtarethebalance.
3.PrewetthenPipetteroomtemperaturereagentgrade
waterintothetipusinggoodpipettingtechnique.
4.Dispensethegradewaterontotheweighboat.
5.Allowthebalancetoequilibrateandrecordthe
reading.
6.Repeatsteps10–20times(dependingonthecriteria
forprecisionestablishedbyyourlaboratory).

10. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Procedure
Dr. TamerSoliman
Foramulti-channelPipette
•Itisimportanttocheckeachchannel,sincethey
candevelopproblemsindependently.
•Apracticalprocedurewouldbetoverifyonechannel,
using10datapoints,ateachof3differentvolume
settings.Thenverifythattheotherchannelsare
performingproperly,byusingfewerdatapointsat
oneortwovolumesettings.

11. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Dr. TamerSoliman
Density of Water at 20ºC = 0.998
at 25ºC = 0.997
at 30ºC = 0.996
(pipetted) water Volume =
measuredwaterWeight
(using Analytic balance)
waterDensity (sp. gravity)
(at the measured room temperature
and atmospheric pr.)
Formula

12. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Dr. TamerSoliman
Precision
•Precisionisameasureofhowclosetogethera
numberofvaluesare.Theclosertogethertheyare,
thebettertheprecision.
•Imprecisionistheoppositemeaningofprecision.
•Imprecisioniseither:
oAbsoluteImprecision
expressedasSD(standarddeviation)of10samples.
oRelativeImprecision
expressedasCV(coefficientofvariation)of10samples.
SD =Σ ( x
i–x̄)
2
( n –1)
CV =
SD
x̄(mean of measured pipetted volumes)

13. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
Calculation
Dr. TamerSoliman
Accuracy
•Accuracyishowclosearesultcomestothe"true"
value
•Inaccuracyistheoppositemeaningofaccuracy.
•Inaccuracyisexpressedasthedeviationofthemean
of10samplesfromthesetpointvolume.
•Inaccuracycanbeexpressedineither:
•Absoluteunitssuchasmicroliters,or
•Relativeunitssuchaspercent.
Set Volume–Mean of pipetted volumes
Inaccuracy =
Set Volume
Accuracy=
Mean of pipetted volumes
Set Volume
ةبسنطخلاأ
x100

14. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
MicropipettCalibrationWorksheet
Dr. TamerSoliman
Date:
Accuracy Range: + %
Allowable Precision: Actual Precision:
Calculated Accuracy: %
Acceptable?
Acceptable?
Yes or No
Micropipette ID:
Ambient Temperature: H
2O Density:
Volume: µl
Pipette readings Difference from the mean (Difference)
2
(x
i
) (x
i
–x̄) (x
i
–x̄)
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Sum (Σ)
Mean(x̄)

15. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
MicropipettCalibrationWorksheet
Dr. TamerSoliman
Worksheet for Variable Micropipette (20 –200 µl) Calibration
[ At Water Density (specific Gravity) = 1.000]
At 20 µl setting
(Low Volume Setting)
At 100 µl setting
(Mid Volume Setting)
At 200 µl setting
(High Volume Setting)
WeightVolume WeightVolume WeightVolume
1 0.0198 g19.8 µl 1 0.0989 g98.9 µl 1 0.1995 g199.5 µl
2 0.0197 g19.8 µl 2 0.0988 g98.8 µl 2 0.1992 g199.2 µl
3 0.0199 g19.8 µl 3 0.0991 g99.1 µl 3 0.1989 g198.9 µl
4 0.0199 g19.8 µl 4 0.0987 g98.7 µl 4 0.1995 g199.5 µl
5 0.0197 g19.8 µl 5 0.0990 g99.0 µl 5 0.1993 g199.3 µl
6 0.0198 g19.8 µl 6 0.0989 g98.9 µl 6 0.1995 g199.5 µl
7 0.0197 g19.8 µl 7 0.0988 g98.8 µl 7 0.1994 g199.4 µl
8 0.0199 g19.8 µl 8 0.0991 g99.1 µl 8 0.1995 g199.5 µl
9 0.0199 g19.8 µl 9 0.0987 g98.7 µl 9 0.1990 g199.0 µl
10 0.0197 g19.8 µl 10 0.0990 g99.0 µl 10 0.1991 g199.1 µl
Sum (Σ)0.1980 g198.0 µlSum (Σ)0.9890 g98.9 µlSum (Σ)1.9920 g1992.0 µl
Mean 0.0198 g19.8 µlMean0.9890 g98.9 µlMean 0.1992 g199.2 µl

16. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
ProceduralNotes
Dr. TamerSoliman
•Avoidhandlingpipettetipswithyourfingers.
Usealintfreewipeorforcepswheneverpossible.
•Tworeadingsarerequired.Twofreshpipettetips
mustbeusedtoconfirmaccuracy.
•PeanutOilcanbeusedtopreventevaporationof
water.Simplyplaceasmallamountoftheoilinthe
weighboatinproceduralstep2.
•Oneweighboatmaybeusedformultiplereadings
withatareperformedbetweeneachweight
measurement.Multiplepipettecalibrationswill
requiretheboattobeemptiedandre-zeroed.
Reminder:Thebalancewillnotallowthetareofa
negativereading.Thebalancemustbere-zeroed.

17. How To Calibrate Your Micropipette?
Generic Procedure for the
Calibration of Micropipettes
using the Gravimetric Method
InterpretationofResults
Dr. TamerSoliman
1.Pipettesnotmeetingtheestablishedacceptance
criteriaforaccuracyorprecisionshouldreceive
routinemaintenancebeforebeingcalibrated
again.
2.Ifapipettecannotmeettheacceptancecriteriait
shouldberemovedfromserviceuntilitcanbe
servicedbythemanufactureroranapproved
vendor.
3.Pipettesthatdomeettheestablishedacceptance
rangesshouldbelabeledwithastickerthat
recordstheactualaccuracyofthepipette,the
dateofcalibrationandtheinitials.