METROLOGY 101

Calibrating a Micropipette
Martin de Groot
Kelvin Training

This paper discusses the calibration of pipette, with particular attention to the type that is used and calibrated most
frequently. This is the so-called air-displacement pipette, single channel, adjustable volume pipet. The new ISO/IEC 17025,
published November 2017, demands more attention to the agreement of the calibration procedure with the customer. The
paper describes points that need to be agreed with the customer and calibration by the gravimetric method (weighing).
The alternative calorimetric method (ISO 8655-7) is not described in this paper.

The Use of Micropipettes The calibration procedure must reproduce the method
of use as much as possible. For the gravimetric approach,
A good explanation of the do’s and don’ts when using a volume of water is added to a vessel on a balance. This
the pipette is shown in a 10 minute YouTube film of the volume is weighed and the weight is converted to volume
University of Leicester [1] (see Figure 1). through the known density of the reference weight(s) used
There is a significant difference in use of these pipettes to calibrate the balance and the equations of the densities
by either the forward or reverse method. Figure 2 shows of water and air. This correction factor is called the Z-factor
the difference between the two approaches. as defined in ISO 8655-6 [2].
It is generally recommended that tips of the original
supplier should be used with the pipette. Alternative tips Method and Points of Attention
can only be used after sufficient experience to ensure the
tip fits the particular pipette type. The tip shape, surface The new ISO/IEC 17025:2017 is even more particular than
roughness, and wettability (adhesion) are of influence the 2005 version about the prior arrangements to be fixed
on the amount of water taken in by the pipette. Take with the customer. Calibrations need to meet the customer
appropriate care in attaching the tip on the pipette. There requirements whenever possible. There are a few items to
must be a close seal between pipette and tip that can easily observe when calibrating pipettes.
be misfit by rough treatment. Tips are for single use only. Calibrate the pipette in the same way as it is used: if the
Make sure that you are not contaminating the pipette customer uses forward (Figure 2) pipetting, the pipette
body with the liquids that you take in. Immerse the pipette must be calibrated that way too. It might be that your
tip 2 mm to 3 mm in the liquid to be taken in. Operate the customer wants to have the calibration data expressed at
pipette with care and (for variable volume pipettes) always a nominal temperature of 20 °C as described in [6].
set the volume from higher to lower volume to avoid that
the measurement reproducibility is influenced by the Maintenance
hysteresis of the pipette mechanism. Pipettes are sensitive to wear. Pipette maintenance is a
regular part of the services provided by a calibration lab;
for this, use original parts supplied by the manufacturer.
If replacement of O-ring, seal, or other components is
required, ISO/IEC 17025:2005 (clause 5.10.4.3) only requires
pre-maintenance or pre-adjustment calibration results when
the pipette is to be used for calibration. ISO/IEC 17025:2017
(7.8.4.1d) is more particular and requires pre-adjustment and
repair data when available for all instruments (i.e., including
those not to be used for calibration). When customers
want to save money by eliminating the pre-adjustment
calibration, the data need not be reported, but the new ISO/
IEC 17025 does require the data to be “readily available.”
Note that ISO/IEC 17025 does not specify that the calibration
procedures before and after adjustment must be the same.
Figure 1. This YouTube film is recommended as a good short
Both procedures need to be agreed upon between customer
introduction to air displacement pipettes and their use [1].
and calibration laboratory (2017 clauses: 7.1.3, 7.2.1.4).

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 METROLOGY 101

Figure 2. Forward versus reverse pipetting. To the left (forward Figure 3. A balance set up for micropipette calibrations with
pipetting): fill the pipette to the first stop and empty it by pushing an evaporation trap to minimize the effect of evaporation on
all the liquid out. To the right (reverse pipetting): fill the pipette to the calibration. The balance is placed on a vibration free table.
the maximum extent and then empty the pipette only by pushing (Courtesy Gilson Netherlands)
to the first stop.

Measurement Volumes and this than the preceding 2005 version. While the old standard
Number of Measurements only requires that you consider the uncertainty when stating
The measurement volumes must be agreed upon with conformity to specification on a certificate, the new standard
the customer. ISO 8655-6:2002 describes a minimum of wants you to understand the level of risk of a false statement
three volumes: the nominal volume, 50 % of the nominal when the measurement is within the uncertainty’s range
volume, and the greatest of 10 % of the nominal volume from the specification limit. Current practice is explained
and the lower limit of the useful volume range. For pipette by ILAC [7] such that you cannot make a statement on
calibrations, the repeatability of the calibration dominates conformity if the measurement is within uncertainty’s reach
the uncertainty budget for most of the pipette ranges. of the specification limit. The new ISO/IEC 17025 allows
The number of repeat measurements at each volume is you to follow your customer’s “decision rule” to reach a
advised to be ten; the coverage factor must be calculated conformity to specification decision. Particularly for low
from the effective degrees of freedom that results from the volume micropipettes, this can solve the problem you might
uncertainty analysis as explained in UKAS M3003 [8] and have experienced when a decisive conclusion on conformity
other basic documents on uncertainty analysis. In order to is not possible because the uncertainty is about as large as
minimize the effect from hysteresis in the volume adjusting or even larger than specified tolerances. This means that not
mechanism of the micropipette, agree with the customer that only do you have to agree on the specification tolerances
the volume is always set in the same direction—preferably with your customer but also the decision rule. For more
from higher volume down to the required volume. information on conformity, read clauses 7.1.3 and 7.8.6 of
the ISO/IEC 17025:2017. Further information on decision
Tip rules can be found in UKAS M3003, annex M [8], and a
Agree on the pipette tip (make and type) to be used document published by BIPM on this subject [9].
during the calibration with the customer. Use the same
pipette tip that the customer uses. Besides possible leakage Equipment
when the pipette tip does not properly fit on the pipette, the
pipette tip material and surface roughness have significant For proper gravimetric calibration of pipettes, you need a
influence on the volume of water taken in by the pipette. balance placed on a stable weighing table, a thermometer to
measure water temperature, a hygrometer for measurement
Decision Rule of ambient temperature and relative humidity, a barometer,
The decision rule is a new item in ISO/EC 17025:2017 sufficient pipette tips, pure water and a container to contain
and a major revision compared to the 2005 version of this water, a weighing vessel with either a lid or as part of
this standard. This rule refers to the way it is decided if a an evaporation trap system to reduce evaporation influence
calibrated item conforms to a defined specification or not. on the measurement, and a timer to measure the time for
The new ISO/IEC 17025 is much more specific in requiring evaporation measurement.

Cal Lab: The International Journal of Metrology 20 Jan • Feb • Mar 2018
 METROLOGY 101

Balance pieces ranging from 1 mg to 100 g. Not all weights in this

For calibration of a pipette, you need an accurate set need to be calibrated. You could use two calibrated
(analytical) balance that must be levelled prior to the reference weights for consistency, but also if only one
measurement and placed on a vibration free bench or of the reference weights is calibrated, the linearity can
table. The balance must have resolution of 10  µg for be derived from differential measurements.
pipettes between 10 µl and 100 µl and 1 µg for pipettes For this, add the weights on the weighing pan in the
with smaller volumes. Larger volumes can be calibrated same order as you remove them. This order may be: 1 mg,
with 0.1  mg balances. For best uncertainties, the 2 mg, 10 mg, 20 mg, 100 mg, 1 g, etc. The added mass for
calibration of the balance requires particular attention to each weight should correspond with the reduced mass
the linearity of the balance in its lowest ranges. After all, when you remove the same weight. Note that the weight
when using the balance, you use the balance to measure is not added at the same load as when it is removed.
volumes between 1 µl (approximately 1 mg) up to, for Comparing the mass changes for each weight shows
example, 100 µl (100 mg on your balance). The balance the nonlinearity of the balance (see Figure 4). For this
you use for this calibration will have a maximum reading measurement, not all weights need to be calibrated, as
of some 5 grams. Make sure you know the linearity of stability is the important feature that this method uses.
the balance over the lowest milligrams as well, as that Figure 4 shows that at higher loads, the calibration of
is where you are actually using the balance! Table 4.1 of the weights become an important contributor to the bias
ISO 8655-6 [2] gives the balance requirements. (difference between reading and calibrated value of the
reference weight). This effect drops out if you compare
Balance Calibration the measured mass differences rather than using the
Calibration of the balance may be done by an external bias. The reference weight(s) fix(es) the traceability of
service agent or by yourself. The balance uncertainty your balance, while the linearity measurements relate
contributes quite (if not most) significantly to the the other scale readings to the bias of the reference
calibration of a pipette at lower volumes (typically weight. The bias is larger than the non-linearity due to
below 100 µl). the uncertainties of the class E2-weights that drop out
If you calibrate the balance yourself, use a set of mass in the subtractions explained in Figure 4.

Figure 4. An analytical balance (total range 215 g, resolution 10 µg) was calibrated adding
1 mg, 20 mg and 200 mg weights (m) to a balance loaded with 20 g, 100 g and 150 g (L).
The bias is equal to the balance reading minus the weight certificate values. For an ideal
balance, the subtraction of the balance reading after and before adding a weight should
be independent of the prior load. Differences δm between these subtractions for different
loads are caused by balance nonlinearity. This nonlinearity is shown both in terms of the
Load, L, and in terms of the applied weights, m.

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 METROLOGY 101

Ambient Conditions to measure.

You shall need two thermometers: one to measure air A measurement cycle consists of 10 measurements;
temperature and one for the water temperature of the if the customer prefers (to cut cost of calibration) less
water in the container containing the source water. The measurements per volume, the uncertainty will be larger
water temperature measurement is the most demanding on because of the required correction of the coverage factor.
accuracy of measurement and contributes more significantly Tare the balance after the measurement and before adding
than the other ambient condition parameters. The a new filling in the weighing vessel.
thermometer may not be so heavy that it makes the water Take notice of the way that water is to be taken in the pipette
container fall over. The thermometer should be calibrated to and to be delivered in the weighing vessel as described in [2
within 0.1 °C uncertainty. The air thermometer is to measure section 7.2]. In Germany, DAkkS requires a slightly deviating
the difference between air and water temperature and must approach for this [10 sections 7.2 and 7.3]. While ISO 8655-
also be calibrated to within 0.1 °C. 6 [2] requires a new tip for every measurement, Guideline
The relative humidity must be measured even though it DKD-R 8-1 “Calibration of piston-operated pipettes with air
has no direct influence on the analysis. The humidity does cushion” [10] allows, under conditions, that the same pipette
influence the evaporation in the weighing vessel on the tip may be used for the full measurement cycle. It is advised
balance. The evaporation causes the weight on the balance to to at least replace the tip for each different volume. Pre-wet
drift. This can be minimized by using a lid on the weighing a newly fitted tip once after replacement.
vessel after adding each volume of water or by using an For evaporation measurements, the time is to be measured
evaporation trap. The ambient humidity must be higher than for a measurement cycle. Then over this time, measure the
50 %rh and must be measured to within an uncertainty of drift on the balance due to evaporation. Divide this by ten
5 %rh. For measurement of the evaporation, and allowing to get the evaporation for a single measurement. Repeat this
a correction and/or the evaluation of its uncertainty, a timer measurement a number of times to allow for an uncertainty
is needed. This timing device should have an uncertainty estimate of this effect.
of 1 s or better.
The ambient pressure must be measured with a Z-factor and Pipette Calibration Data
barometer with a resolution of 1 hPa and an uncertainty
of better than 5 hPa. The analysis of a pipette calibration is not a straightforward
calculation. The widely-used gravimetric calibration
Calibration Procedure method (described in ISO8655-6 [2]) relies on an amount of
pure water to be transferred into the vessel placed on the
As part of the preparation, make sure both pipettes and balance. The weight of this water relates to the volume of the
tips, water, and all associated measurement equipment are transferred water by a formula that incorporates the density
sufficiently climatized prior to the measurement. of the water, density of air and the buoyancy effect affecting
Calibrate the balance once per day using a calibrated the weight of the water on the balance. This formula is, in
weight and adjust the balance using its internal adjustment practice, replaced by the so-called Z-factor, that depends
system regularly to make sure the balance is corrected for on water temperature, air temperature, and pressure and
ambient temperature drift. Make sure the balance setting can be found in table A1 of ISO 8655-6. ISO 8655 takes the
does not automatically do this, ruining your measurement air temperature to be equal to the water temperature. For
in the middle of a measurement cycle. Also verify that the more detail, see Figure 5 and accompanying text.
drift setting of the balance is off.
Verify that the difference between water temperature Uncertainty Analysis
and ambient temperature is not bigger than 0.2 °C. During
the calibration, the temperature should not drift by more Z-factor Uncertainty
than 0.5 °C. For proper uncertainty analysis the sensitivity of the
At the beginning of the measurement cycle at each volume, Z-factor to the influence parameters (temperatures,
a tip shall [2] be used only to pre-wet the air inside the pressure, but also humidity) is to be calculated. This
pipette. For this, fill the pipette with water from the reservoir can be done analytically as in ISO/TR 20461 [6] but also
five times and empty the water to waste. This will more or by numerical variation of the influence parameters. The
less “saturate” the air inside the pipet so that evaporation uncertainty basics are laid out in ISO/TR 20461.  
during the calibration is minimized. Evaporation would As stated before, the uncertainty from relative humidity
cause a significant increase in volume of the air cushion of is negligible. The Z-factor dependence can be simply
the pipette, displacing the water from the pipette tip. Before found by looking at the Z-dependence on pressure or
the calibration, remove the tip and replace it by a new one temperature in table A.1 in ISO 8655 [2]. These so-called
that you now carefully fill only once emptying this also to sensitivity factors are used to multiply the thermometer and
waste. Tare the balance. It is only now that you are ready barometer uncertainties in the budget for the calculation

Cal Lab: The International Journal of Metrology 22 Jan • Feb • Mar 2018
 METROLOGY 101 Buoyancy Effect and the Z-factor
When using an ordinary (analytical) balance for pipette
of the uncertainty contributions from these ambient calibration, the balance was calibrated by a stainless
measurements on the calibrated volume. For more accurate steel reference weight with density ρr = 8000 kg m-3. This
calculation of the uncertainty the ISO/TR 20461 approach density is a fixed value within the required accuracy.
is better calculating separate sensitivity coefficients for During pipette calibration the balance weighs water with
ambient temperature and water temperature. a density of approximately 1000 kg m-3 and compares this
ISO/TR 20461 explains the temperature dependence of with the weight of the stainless steel reference weight
the pipette and includes an uncertainty component in the previously used for the calibration. The density of water
budget for the heating of the pipette by the operator. depends on the temperature of the water [3].

Balance Uncertainty
The calibration of the balance results in two components
for the uncertainty. One is the linearity component the other
is the uncertainty following from the calibration using the
reference weight.
Smaller contributions are given as the reproducibility
of the balance and the readability of the balance. It can be
argued, however, that these components are already part
of the balance calibration. As these are smaller than the
balance calibration contribution, these components have
no significant influence in the combined uncertainty.
Temperature drift sensitivity of the balance results in Figure 5. The upwards buoyancy effect is caused by the weight of
another generally small contribution per degree ambient displaced air by the volume of the stainless steel weight (on the left)
vs. the larger amount of displaced air by the volume of water on the
temperature instability. This can be calculated using the
right. The weight on both loads is partly compensated by the weight
maximum allowed temperature deviation being 0.5 °C. of the displaced air.

Water Purity The upwards buoyancy effect is caused by the

The water purity can be verified by boiling out a weighed weight of displaced air by the volume of the stainless
volume of water and weighing the residual. As very pure steel weight (on the left) versus the larger amount of
(distilled) water should be used, the uncertainty from this displaced air by the volume of water on the right. The
effect should be negligible. weight on both loads is partly compensated by the
weight of the displaced air. The water having larger
volume for the same weight experiences larger upward
Repeatability force because of the larger volume of displaced air. The
Every measurement is repeated a number, n, of times
air density is approximately 1.0  kg  m-3 and depends
with n normally being 10 [2]. The repeatability is calculated
on air temperature, pressure, and to a lesser extent, on
from the standard deviation s. This standard deviation can relative humidity; the equation for this can be found in
be compared with the required value in the specification. [4]. Part of the equation in reference [4] is the molar gas
The component for repeatability of the measurement in constant. This value has very recently undergone a very
the uncertainty budget is the standard deviation of the small change as a consequence of the redefinition of the
_
mean s​ ​ = s/√n. Generally this component is the dominant SI units. As part of the revision of the SI, new values
contributor to the uncertainty. The operator has a very large have been defined for the Boltzmann constant and the
influence on the calibration result. Avogadro constant. The product of these is equal to the
gas constant. The effect of this change to the Z-factor is
Further Uncertainty Components: however very small and insignificant for the calculation
of the Z-factor [5].
Pressure vs. Altitude
Knowing the density of water ρw (t90,w ) as a function
Thorough German research has shown that this
uncertainty calculation is not complete [11,12]. There are of water temperature, the density of air ρa (t90,a ,p, xv ) as
some other dominant factors contributing to the uncertainty a function of temperature, humidity and pressure, and
that are not sufficiently identified in ISO/TR20461. These the correction factor from buoyancy can be calculated
relate to pressure effects that become significant when from [6]: ​ρ​​− ​ρ​ ​
working with the pipette on a different altitude than where ​  ​ρ1​ ​ ​ × ______
Z( ​t ​90,w​, ​t ​90,a​, p, ​x v​ ​) = __ ​  ​ρ​r ​− ​ρa​ 
​ ​ 
r w a
the pipette was calibrated. DKD document [10] describes
an elegant formula (3) allowing correction of a calibration As the dependence of this Z-factor from humidity is
result to a different pressure. An uncertainty component of insignificant in comparison with the other parameter
20 hPa is advised on the Z-factor to account for this effect. dependencies, the humidity dependence is neglected.

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 METROLOGY 101

Figure 6. CMC example uncertainty based on a combination of relevant terms from ISO/TR 20461 and DKD R8-1 (see Table 1
on the following page).

Figure 7. Uncertainty budget for a volume corresponding with the CMC budget in Figure 6.

Cal Lab: The International Journal of Metrology 24 Jan • Feb • Mar 2018
 METROLOGY 101

U Expanded uncertainty at 95 % percent coverage probability.  References

u(<m>meas) Repeatability of measurement specification ISO 8655-2. [1] University of Leicester, 25 Jun
2009, "Using a Micropipette,"
u(δDKD,handling) DKD handling effect 0.07 % of volume.
https://youtu.be/uEy_NGDfo_8.
u(RHDKD) DKD relative humidity effect as described in text for 5 %rh uncertainty. [2] ISO 8655-6; 2002. Piston-oper-
u(δtwater_air) Difference between air and water temperature at 0.2 °C.
ated volumetric apparatus—Part 6:
Gravimetric methods for the determi-
u(PDKD) DKD pressure effect at 0.01 % of volume per 1 %rh. nation of measurement error.
u(ρwater) Density of water uncertainty estimated as 0.005%. [3] M. Tanaka, et al., "Recom-
mended table for the density of
u(Zt-water) Z-factor water temperature at uncertainty of 0.2 °C.
water between 0 °C and 40 °C
u(δmbalance) Balance calibration 0.01 mg for 5 g balance and 0.15 mg for other balance. based on recent experimental re-
u(ZP) Z-factor pressure contribution at uncertainty of 10 hPa. ports," Metrologia, 2001, 38, 301-
309
u(Zt-air) Z-factor air temperature at uncertainty of 0.5 °C.
[4] A. Picard, et al., "Revised for-
u(ZRH) Z-factor relative humidity contribution at uncertainty of 10 %rh. mula for the density of moist air"
(CIPM-2007), Metrologia 2008, 45,
u(δevap) Evaporation: 4 nl uncertainty in measured effect.
149155.
u(δt,balance) Temperature sensitivity of balance for standard balance specification. [5] D.B. Newell et. al., "The CO-
DATA 2017 values of h, e, k, and
Table 1. UNCERTAINTY BUDGET components used to produce Figures 6 and 7. NA for the revision of the SI,"
Metrologia 2018, 55 L13-L16.
The DKD document [11] also explains that this pressure [6] ISO/TR 20461:2000 (&Cor 1: 2008, IDT) Determination
effect depends on humidity. It is argued that while of uncertainty for volume measurements made using the
transferring the water from the container to the weighing gravimetric method. (Note the correction document of
vessel, water evaporates from the aspirated water into the 2008 specifying wrong signs of some coefficients in the
air cushion. This increases the volume of and humidity in original document!)
the air cushion and drives some of the liquid out of the [7] ILAC G8:03/2009 Guidelines on the Reporting of Compli-
pipette. An uncertainty contribution has been taken from ance with Specification.
studies to amount to 0.007 % of the transferred volume [8] United Kingdom Accreditation Service, The Expression
per change of humidity by 1 %rh for 100 µl and 1000 µl of Uncertainty and Confidence in Measurement, M3003
nominal pipette volumes. At 1 µl nominal pipette volume, Ed. 3, November 2012.
this effect is 0.01 % of volume per change of humidity by [9] JCGM 106:2012 Evaluation of measurement data – The
1 %rh. Prewetting to increase humidity inside the pipette role of measurement uncertainty in conformity assessment,
becomes even more important. BIPM 2012.
[10] PTB/DKD Guideline DKD-R 8-1 Calibration of piston-
Handling Component operated pipettes with air cushion, 12/2011.
 DKD document [10] recognizes handling influences on [11] K. H. Lochner, R. Feldmann, J. Pfohl, PTB/DKD Expert
the calibration through: report DKD-E 8-2 Analysis of influencing parameters on
1. mechanical effects (hysteresis reproducibility of the calibration of piston-operated pipettes with air cushions, Is-
piston stroke); sue 05/2013.
2. influences by the operator (waiting times, pace of
[12] C. Spälti, M. Polin, S. Finkbeiner, PTB/DKD DKD-E
work, pipette angle, operating force, immersion
8-3  Einfluss der Höhenlage auf das Volumenergebnis einer
depth); and
KolbenHubpipette mit Luftpolster,  Ausgabe 09/2013.
3. hand warmth, as the thermometer is heated in the
hand of the operator.
To account for these effects, German accredited
laboratories are required to include a contribution of 0.1% Martin de Groot ([email protected]), Kelvin
of the calibrated volume in the budget for handling. Training, Kampdijklaan 4, 5263 CJ Vught, Netherlands,
  https://kelvin.training/.
For readers wanting to do some calculations themselves,
a GUM workbench example budget is available from our
website (Kelvin.training).

Jan • Feb • Mar 2018 25 Cal Lab: The International Journal of Metrology