Pipettes

Different types of pipettes and their uses

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Pipets
Tauqeer Ali

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ContentsContents
ClassificationClassification
Pipetting techniquePipetting technique
Pipet termsPipet terms
MicropipetMicropipet
Micropipet typesMicropipet types
Advantages Advantages
Forward PipettingForward Pipetting
Reverse pipetting Reverse pipetting
Risk factorsRisk factors
Preventing cross-contaminationPreventing cross-contamination
Tip SelectionTip Selection
Criteria for selection of pipetCriteria for selection of pipet
Quality controlQuality control

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PipetPipet
Utensil to transfer liquidUtensil to transfer liquid
Class A materialClass A material
20ml or less 20ml or less
Accurate measurement accurate resultsAccurate measurement accurate results

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ClassificationClassification
1.1.Manual PipetsManual Pipets
2.2.Mechanical devicesMechanical devices
Manual Pipets are Manual Pipets are
1.1.Transfer pipet (volumetric)Transfer pipet (volumetric)
2.2.Measuring pipetMeasuring pipet
3.3.MicropipetMicropipet
subclassificationsubclassification
TCTC TDTD TD/Blow outTD/Blow out

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TC (Rinse-out)TC (Rinse-out)
Contain exact amountContain exact amount
Accurate measurementAccurate measurement
Must be completely drainMust be completely drain
Rinsed with solventRinsed with solvent
E.g. E.g.
Sahli hemoglobinSahli hemoglobin
Transfer microTransfer micro

Lang-LeavyLang-Leavy

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TD/BLOW-OUTTD/BLOW-OUT
Deliver exact volume Deliver exact volume
Allowed to drainAllowed to drain
Than blow outThan blow out
Not rinsed outNot rinsed out
Etched ring / frosted endsEtched ring / frosted ends
E.g.E.g.
Ostwald-folinOstwald-folin
SerologicalSerological

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TDTD
Deliver exact volume Deliver exact volume
Allow to drain by gravityAllow to drain by gravity
No blow outNo blow out
E.g.E.g.
Volumetric transferVolumetric transfer
MohrMohr

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Basic classification Basic classification
Measuring pipetMeasuring pipet
Glass tube Glass tube
Graduated Graduated
Transfer pipetTransfer pipet
Cylindrical bulbCylindrical bulb
Narrow tube at both endNarrow tube at both end
Tapered tipTapered tip
MeasuringMeasuring TransferTransfer
SerologicSerologic volumetricvolumetric
Mohr Mohr Ostwald-FolinOstwald-Folin
BacteriologicBacteriologic PasteurPasteur

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Serological pipetSerological pipet
Long glass or plastic tubeLong glass or plastic tube
Uniform diameterUniform diameter
Tapered tipsTapered tips
Graduated till tipGraduated till tip
Blow outBlow out
Variable openingsVariable openings
Large opening_ viscous fluidsLarge opening_ viscous fluids

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Mohr pipetMohr pipet
Uniform diameterUniform diameter
Tapered delivery tipTapered delivery tip
Graduations incised on stem onlyGraduations incised on stem only
Self draining Self draining
Long tipsLong tips

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Bacteriologic pipet
Glass or polypropylene
Use for transport of bacterial
specimen esp. pus
Sealed by flame and cotton plug
Provide maximum sterility

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Volumetric pipetVolumetric pipet
Long glass tubeLong glass tube
Open ended bulbOpen ended bulb
Max line limit Max line limit
Single volumeSingle volume
Tapered delivery tipTapered delivery tip
For dilute aqueous SolutionFor dilute aqueous Solution
Self draining Self draining
Reliability decrease as sizeReliability decrease as size

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Ostwald-Folin pipet
Bulb closer to delivery tip
Viscous fluids
Blow out
Opaque fluid read top of meniscus

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Pasteur pipetPasteur pipet
Don’t have calibration marksDon’t have calibration marks
Transfer fluidsTransfer fluids
Without consideration of volumeWithout consideration of volume
One time use One time use
Plastic or glassPlastic or glass
Beral pipetBeral pipet
flexible soft plastic,has a built-in bulb on the end.flexible soft plastic,has a built-in bulb on the end.

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Pipetting technique
Pipetting bulb
Held vertical
Read at eye level
Lowest part of
meniscus at line level
Unrestricted flow
Inclined container
and touch tip
Etched ring or
frosted end
Blow out after drain

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MicropipetsMicropipets
Semiautomatic devices Semiautomatic devices
≤ ≤ 1ml1ml
Mostly are TC (rinsed out)Mostly are TC (rinsed out)
1-1000µl1-1000µl
0.5µl-20ml0.5µl-20ml
Fixed or adjustableFixed or adjustable

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Pipet termsPipet terms
AdjustmentAdjustment - altering the pipet so that the dispensed - altering the pipet so that the dispensed
volume is within the specifications.volume is within the specifications.
AspirateAspirate - to draw up the sample.- to draw up the sample.
Dispense/distributionDispense/distribution - to deliver the sample.- to deliver the sample.
Calibration checkCalibration check - checking the difference between - checking the difference between
the dispensed volume and the selected volume.the dispensed volume and the selected volume.
AccuracyAccuracy -- is the degree of closeness of volume is the degree of closeness of volume
indicated and dispensed. indicated and dispensed.
PrecisionPrecision – reproducibility the degree to which further – reproducibility the degree to which further
measurements show the same or similar results.measurements show the same or similar results.
Purge – blow outPurge – blow out

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Advantages
Adjustable
Ergonomic design
Multiple wells
Piston driven
Stability
Safety
Ease of use
Disposable tips
No washing or drying
Save time
Avoid cross contamination
Improve precision

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Micropipets typesMicropipets types
1.1.Air displacementAir displacement
2.2.Positive displacementPositive displacement
3.3.Dilutor or dispenserDilutor or dispenser

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Air displacement pipet Air displacement pipet
Accurate & preciseAccurate & precise
Relies on piston for suction creationRelies on piston for suction creation
Air cushion between piston and disposable tipAir cushion between piston and disposable tip
No contact of piston and sampleNo contact of piston and sample
Can be used asCan be used as
Forward PipettingForward Pipetting
Reverse Pipetting Reverse Pipetting

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Forward PipettingForward Pipetting
Preparation Preparation
Hold in vertical position. Hold in vertical position.
Depress the plunger to first stop position.Depress the plunger to first stop position.
Aspiration Aspiration
Immerse tip in the liquid.Immerse tip in the liquid.
move plunger smoothly to the rest position. move plunger smoothly to the rest position.
Distribution Distribution
Place tip at an angle (10 to 45°) against the inside wall of the receiving vessel. Place tip at an angle (10 to 45°) against the inside wall of the receiving vessel.
Depress plunger to the first stop position.Depress plunger to the first stop position.
Purge Purge
depress the plunger to the second stop position (“blow-out” )depress the plunger to the second stop position (“blow-out” )
Remove pipette tip end from sidewall by sliding it up the wall.Remove pipette tip end from sidewall by sliding it up the wall.
HomeHome
 Allow the plunger to move up to the rest position.Allow the plunger to move up to the rest position.

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Reverse PipettingReverse Pipetting
PreparationPreparation
Hold vertical position. Hold vertical position.
Depress the plunger to the second stop position.Depress the plunger to the second stop position.
Aspiration Aspiration
Immerse the pipette tip in the liquid.Immerse the pipette tip in the liquid.
Allow the plunger to move up smoothly to the rest position.Allow the plunger to move up smoothly to the rest position.
DistributionDistribution
Place tip at an angle (10 to 45°) against the inside wall of the receiving vessel. Place tip at an angle (10 to 45°) against the inside wall of the receiving vessel.
Depress the plunger to the first stop position. Wait one second.Depress the plunger to the first stop position. Wait one second.
Re-Aspiration Re-Aspiration
pipette tip is to be reused for the same sample.pipette tip is to be reused for the same sample.
 maintain the plunger in the intermediate position for subsequent immersion for the next maintain the plunger in the intermediate position for subsequent immersion for the next
pipetting cycle and restart operation 2. pipetting cycle and restart operation 2.
Complete purge Complete purge
Wait one second and purge. Wait one second and purge.
If the pipette tip is not to be re-used, depress the plunger to purge position over an If the pipette tip is not to be re-used, depress the plunger to purge position over an
appropriate waste container and then eject the tip.appropriate waste container and then eject the tip.

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Positive displacement pipetPositive displacement pipet
Moving piston in pipet tipMoving piston in pipet tip
Carry over concernCarry over concern
Rinsing and blotting b/w samplesRinsing and blotting b/w samples

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Dispenser or dilutor
To dispense repeatedly specified
volume
Attach to reagent bottle directly
Depression of plunger dispense
specified volume
Error rate 1%
Precision rate 0.1%
Useful for serial dispensing

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Mechanical devices
Single well or multiple wells
Use disposable tips or washing out
Programmable
96 or 384 wells

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General guidelinesGeneral guidelines
Check at the beginning of work, wipe with 70% ethanol.Check at the beginning of work, wipe with 70% ethanol.
Set the volume.Set the volume.
Fluid and pipet tip on same temp.Fluid and pipet tip on same temp.
Recommended tip for pipet. Recommended tip for pipet.
High-quality tips of contamination-free polypropylene.High-quality tips of contamination-free polypropylene.
Tip for single use.Tip for single use.
Avoid inverting pipette when liquid in the tip.Avoid inverting pipette when liquid in the tip.
Avoid fingers contamination by using the tip ejector and gloves.Avoid fingers contamination by using the tip ejector and gloves.
Use Pipet stands Use Pipet stands
Check calibration regularly.Check calibration regularly.
Follow the instructions for recalibration by the manufacturer. Follow the instructions for recalibration by the manufacturer.

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Risk factorsRisk factors
Repetitive motion and excessive force of the Repetitive motion and excessive force of the
thumb.thumb.
Awkward postures of the hand, wrist, arms and Awkward postures of the hand, wrist, arms and
shoulders.shoulders.
Bending and twisting of the wrist.Bending and twisting of the wrist.
Forceful gripping.Forceful gripping.
Holding the hand in an elevated position and Holding the hand in an elevated position and
distant from the body.distant from the body.
Sustained postures.Sustained postures.

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Preventing cross-contaminationPreventing cross-contamination
Pipet-to-samplePipet-to-sample
contaminated contaminated pipettepipette or or tipstips can cause contamination can cause contamination
of samples.of samples.
Prevention:Prevention:
Use sterilized tips Use sterilized tips
if possible if possible autoclaveautoclave the pipet. the pipet.
Change the tip after pipetting of each sample.Change the tip after pipetting of each sample.
Store the pipet vertically.Store the pipet vertically.

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Sample-to-pipetSample-to-pipet
Two waysTwo ways
Samples can enter the cone of the pipet.Samples can enter the cone of the pipet.
aerosols of sampleaerosols of sample
Prevention:Prevention:
vertical pipettevertical pipette
Release the push-button slowly.Release the push-button slowly.
To avoid aerosol contamination, use filter tips or use a positive To avoid aerosol contamination, use filter tips or use a positive
displacementdisplacement
Sample-to-sample (carry-over)Sample-to-sample (carry-over)
The remains of sample A can mix with next sample B.The remains of sample A can mix with next sample B.
Prevention:Prevention:
Change the tip after each sample.Change the tip after each sample.
autoclave or cleaning of pipetteautoclave or cleaning of pipette

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Tip SelectionTip Selection
Use recommended tips onlyUse recommended tips only
High quality pure polypropyleneHigh quality pure polypropylene
Free from dust or particlesFree from dust or particles
Uniform in size and shapeUniform in size and shape
Accurately centered for tip orificeAccurately centered for tip orifice
Free from metals, like cadmiumFree from metals, like cadmium
Securely sealed: There should be good tip cone match for a Securely sealed: There should be good tip cone match for a
perfect seal and tip ejectperfect seal and tip eject
Free from blemishes, flashing or any protrusionsFree from blemishes, flashing or any protrusions
Highly resistant to chemicalsHighly resistant to chemicals
Highly stable thermallyHighly stable thermally

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Criteria for selection of pipetCriteria for selection of pipet
Level of accuracy and precision requiredLevel of accuracy and precision required
How well the device performHow well the device perform
Autoclaved or notAutoclaved or not
Ease of useEase of use
Work loadWork load
Maintenance and calibrationMaintenance and calibration
Cost, consumable and replacement partsCost, consumable and replacement parts
availability availability

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Quality control
•General
•Check accuracy and precision
•Depend amount of use
•At least once or twice per year
•Routine maintenance
•Air displacement
–Stroke length
–Air seal
•Positive displacement
–Spring check
–Replacement of Teflon tip

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Quality control validationQuality control validation
MethodsMethods
Gravimetric Gravimetric
Spectrophotometric Spectrophotometric

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Gravimetric methodGravimetric method
Gravimetric measurement of dispense aliquot Gravimetric measurement of dispense aliquot
of water with density correctionof water with density correction
ProcedureProcedure
1.1.Water, weighing vial & pipet at temp room.Water, weighing vial & pipet at temp room.
2.2.Record temp of pure water.Record temp of pure water.
3.3.Weigh empty stoppered vial. (wWeigh empty stoppered vial. (wvv))
4.4.Dispense sample of water in weighed vial.Dispense sample of water in weighed vial.
5.5.Re-stoppered, reweigh and record.(wRe-stoppered, reweigh and record.(wff))

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Refer to table “true capacity of glass vessels from the weight of contained Refer to table “true capacity of glass vessels from the weight of contained
water when weighed in air” to obtain correction factor(Ft)water when weighed in air” to obtain correction factor(Ft)
Calculate volume delivered(VD) asCalculate volume delivered(VD) as
VD, ml =(wVD, ml =(wff –w –wvv )xFt )xFt
Example for 10ml pipet:Example for 10ml pipet:

wwff =31.9961g =31.9961g

wwvv =22.391g =22.391g
T = 24T = 24°°CC
Ft = 1.003771Ft = 1.003771
VD = 31.9961-22.0391x1.003771VD = 31.9961-22.0391x1.003771
= 9.9945 mL= 9.9945 mL
Deviation or errorDeviation or error
(10-9.9945) x 100 = 0.055%(10-9.9945) x 100 = 0.055%
1010
0.1% error is acceptable.0.1% error is acceptable.

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Spectrophotometric methodSpectrophotometric method
Alternative to gravimetricAlternative to gravimetric
Use compound that absorb lightUse compound that absorb light
p-p-nitrophenolnitrophenol

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Procedure for calibration of 10Procedure for calibration of 10µµL L
pipetpipet
 Reagents requirementReagents requirement
Na0H 0.01 mol/LNa0H 0.01 mol/L
p-nitriphenol 105mg/dLp-nitriphenol 105mg/dL
Preparation of reagentPreparation of reagent
Dissolve 105mg of p-nitriphenol in 100mL deionized waterDissolve 105mg of p-nitriphenol in 100mL deionized water
Preparation of dilutionsPreparation of dilutions
1.1.Reference dilutionReference dilution
fill three 250mL volumetric flasks with 0.01 mol/L NaOH.fill three 250mL volumetric flasks with 0.01 mol/L NaOH.
Than add to each 1.0mL p-nitriphenol using different pipet each time.Than add to each 1.0mL p-nitriphenol using different pipet each time.
2.2.Test dilutionTest dilution
arrange 5 test tubes, using calibrated pipet add 2.5mL NaOH to each.arrange 5 test tubes, using calibrated pipet add 2.5mL NaOH to each.
to each add 10to each add 10µµL L p-nitriphenol using test micropipet.p-nitriphenol using test micropipet.
read absorbance of each reference and test dilution at 401nm in 10mm cuvet.read absorbance of each reference and test dilution at 401nm in 10mm cuvet.

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p-nitriphenol in NaOH give reading 0.550p-nitriphenol in NaOH give reading 0.550
Average the readings of three reference dilution (AAverage the readings of three reference dilution (A11) should be equal to 0.550) should be equal to 0.550
Average the five test dilutions (AAverage the five test dilutions (A22))
CalculationCalculation
VD (VD (µµL) = AL) = A22 x D x V x D x V
AA11
Where Where
D is dilution of test dilution (1/251 here)D is dilution of test dilution (1/251 here)
V is final volume in microliters of test dilution V is final volume in microliters of test dilution
(2510(2510µµL)L)
So if So if
A1 =0.550A1 =0.550
A2 =0.561A2 =0.561
Volume delivered is 10.20Volume delivered is 10.20µµL.L.
Error is 2%.Error is 2%.
Normal capacity is 0.5%-1%.Normal capacity is 0.5%-1%.

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References References
TietzTietz Textbook of Textbook of Clinical ChemistryClinical Chemistry
Clinical Chemistry:Clinical Chemistry:Principles, Procedures, Principles, Procedures,
Correlations: Michael L. Correlations: Michael L. BishopBishop, Janet L. , Janet L.
Duben-Engelkirk, Edward P. Fody Duben-Engelkirk, Edward P. Fody
Clinical ChemistryClinical Chemistry, , MosbyMosby
A Handbook of Clinical PathologyA Handbook of Clinical Pathology By  By
Ganendrakumar Chakravarti, Kalimoy Bhattacharya Ganendrakumar Chakravarti, Kalimoy Bhattacharya
http://www.gilson.com/literature/pipetting.http://www.gilson.com/literature/pipetting.
http://www.bdbiosciences.com/nvCategory.jsp?http://www.bdbiosciences.com/nvCategory.jsp?
action=SELECT&form=formTree_catBean&item=6195action=SELECT&form=formTree_catBean&item=6195
0606

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