Piston pipets with air cushions have long been the
standard equipment in many laboratories, especially
in medical research and the life sciences for the
transfer of liquids in the microliter range. The ease of
handling of these small volumes made possible the
development of many methods in the life sciences,
chemical analysis, and clinical chemistry. In comparison
to glass pipets, piston pipets offer clear advantages.
They enable very rational and efficient operation
and consequently much higher throughput.
When using piston pipets with the adaptable one-way
plastic tip, the dispensed medium only comes
into contact with the tip because of the air cushion
above it. Therefore, pipet contamination (e.g.,
with radioactive or infectious material) is avoided.
Cleaning after use is not necessary as it is with
Multichannel pipets with 4, 8, 12, or 16 channels
permit even faster and more efficient operation. In
particular, the use of multichannel pipets in combination with multiple-well plates significantly reduces
the amount of pipetting processes required, and
allows considerably faster and more efficient use.
Over time, changes such as corrosion of the piston
rods or drying of the piston seals may occur. This has
a significant impact on measurement accuracy. The
higher the number of channels in the multichannel
pipets, the higher is the probability of a defect in one
of the channels. Because these alterations are not
easily recognizable, it is extremely important to
check the multichannel pipets at regular intervals.
It is for this purpose that a series of rules and regulations
exists. According to ISO 9000 and the GLP
regulations,1 all liquid handling instruments such as
piston pipets must be tested, cleaned, serviced, and
calibrated regularly. For medical analysis, only
instruments with a certificate of conformity may be
used for quantitative measurements. Pipet manufacturers
are required to test their products before they
leave the factory. Certified or accredited laboratories
that use pipets must test them or have them tested
regularly, usually at intervals of 6 or 12 months,
depending on usage. This entails considerable time
and cost for large laboratories that commonly use
The requirements for measurement accuracy
(maximum permissible systematic errors and random
errors) for single-channel and multichannel
pipets are described in ISO 8655-2,2 the successor
to DIN 12650.
The uncertainty of measurement, μ, is given by the
μ = # es # + 2s (1)
Where μ is the uncertainty of the measurement; # es #
is the systematic error; and s is the random error
(repeatability standard deviation).
The systematic error of piston pipets is the deviation
of the nominal or selected partial volume from the
mean value of 10 measurements. The random error
of piston pipets is the repeatability standard deviation
of the 10 measurements.
The test is primarily carried out gravimetrically
according to ISO 8655-6,3 since this is the reference
method. The test procedure required by ISO 8655 is
complex: The piston pipet must be conditioned first,
i.e., the tip must be premoistened five times to saturate
the air cushion in the pipet (dead volume) with
moisture.4–6 Then, the pipet is filled by pushing the
piston to the first stop, dipping the tip into water,
and slowly sliding back the piston. The content of
the tip is emptied into a glass tube and immediately
weighed. To avoid evaporation of the water during
weighing, sealed weighing vessels must be used.
Alternatively, evaporation can be greatly reduced by
using evaporation traps (areas with very high humidity).
Correction by calculation is also possible.5,6
According to ISO 8655-6, the tips may be used only
once for the volume check. A new tip must be used
for each measurement. Since the pipet has already
been conditioned, a single premoistening following
tip exchange is sufficient. In total, this measurement,
including tip exchange and premoistening, is carried
out 10 times.
Using single-channel piston pipets with fixed volumes,
these 10 weighings can be done in a few minutes,
whereas testing multichannel pipets requires
considerably more time. At 10 weighings per channel
for testing, the nominal volume of a 12-channel
pipet, 120 weighings are necessary. For piston pipets
with variable volume, which is always the case with
multichannel pipets, the nominal volume, 50% of
the nominal volume, and the lower limit of the useful
volume range or 10% of the nominal volume
(whichever is greater) must be tested. Thus, 360
weighings are necessary in all.
Compared with the testing of a single-channel pipet
with a fixed volume, the time to test a 12-channel
pipet is 36× greater. Thus, the testing of only one
multichannel pipet typically takes 2–3 hr.
Due to the importance of multichannel pipets in the
medical sector as well areas such as the life sciences,
the German Standards Committee (DIN) for
Laboratory Instruments suggested a research project,
the objective of which was the development of a
method for testing multichannel piston pipets that is
fast and in conformance with the norm.
Figure 1 - Overall view of SpeedCal gravimetric testing unit.
Figure 2 - Detailed view of testing unit. Liquid is pipetted
into the filling tubes of the 12 weighing vessels, enclosed by the
At the onset of the research project, different solutions
were analyzed with regard to time. The greatest time
savings occurred when pipetting with all 12 channels
simultaneously and immediately weighing the dispensed
fluid volume simultaneously. For this purpose, a multichannel
weighing instrument specifically for pipet testing
had to be developed. The SpeedCal test instrument,
shown in Figure 1 (produced by Fraunhofer-Institute for
Silicate Research, Wertheim, Germany, in cooperation
with Sartorius AG, Göttingen, Germany) reduces the
cost of pipet testing while enabling easy, fast, and reliable
testing in accordance with applicable standards.
The instrument can test up to 12 pipet channels simultaneously.
The weighing vessels are enclosed in an evaporation
trap (Figure 2) to minimize measurement error
due to evaporation. Each chamber has a volume of 12 mL; thus several pipets can be tested without having to
empty the chambers between tests. The multichannel
weighing instrument is automatically tared after each
measurement. Once the weighing vessels are filled up,
the operator can continue testing without interruption
by simply suctioning off the liquid with a special device.
With a 12-channel pipet, for example, simultaneous
testing of channels reduces the actual testing time for
360 measurements to 10–12 min, depending on the
operator's level of pipetting experience. The control of
the test procedure and evaluation of results are carried
out by a computer connected with SpeedCal. To convert
the weighing result to the volume, the air pressure,
air temperature, and water temperature must be entered
prior to testing. Following each pipetting, at the touch
of a button the measured weights of the delivered volumes
are read after the settling of the weighing cells
(approx. 10 sec), and are automatically converted into
the corresponding volume.
3 - Original printout of a test record using SpeedCal. Testing of a 12-channel pipet, nominal volume 300 μL, setting 150 μL. All 120 measured values
are shown (in μL). In addition to the mean value and standard deviation taken from all measurements, the mean value and standard deviation of each channel and
each repeat measurement are also shown. It took about 4 min to perform these 120 measurements, including the evaluation and printout. The same period of time is
required for testing at the other two volume settings; thus it takes about 12 min to complete the entire test series of a 12-channel pipet (equal to 360 measurements).
A record of the measurements (Figure 3) is printed
automatically. In addition to the individual values,
this test record includes mean values and standard
deviations for each measurement, i.e., the mean and
standard deviation among the 12 channels, as well as
for each channel—in other words, the mean and
standard deviation over the entire
test series. Furthermore, the record
shows the prevailing ambient conditions,
such as temperature, air
pressure, density of air and water,
and the resulting conversion factor
Z for deriving the volume from the
gravimetric tests. Gravimetric testing
with SpeedCal is fully compliant
with ISO 8655 and DIN 12650,
and delivers results identical to
those obtained with conventional
gravimetric methods (Figure 4).
Figure 4 - Comparison of the results achieved with SpeedCal and the traditional gravimetric
method. A 12-channel pipet with a nominal volume of 300 μL was tested at a setting
of 150 μL. The diagram shows the systematic error and two times the random error, as well
as the sums of these values, which represent the uncertainty of measurement for the pipet.
With SpeedCal, the time needed to
test single-channel pipets is reduced
as well. The 12 vessels allow the
testing of single-channel pipets
without having to wait until the
balances are stationary. This takes
up to 50% less time than is required
for conventional testing of single-channel
With its significantly faster operation
and automatic documentation
features, SpeedCal is well suited not
only for calibration laboratories and
pipet manufacturers, but also for
any laboratory that tests large numbers
of multichannel and single-channel
pipets on a regular basis.
- OECD principles of good laboratory
practice. OECD guidelines for testing
of chemicals. Paris, France:
- Norm ISO 8655-2, 2002. Piston-operated
volumetric apparatus. Part 2:
- Norm ISO 8655-6, 2002. Piston-operated
volumetric apparatus. Part 6:
gravimetric methods for the determination
of measurement error.
- Zeman GH, Mathewson NS.
Necessity of prerinsing disposable
polypropylene pipet tips. Clin Chem
- Michel F, Sommer K, Spieweck F.
Investigations for the determination of
uncertainty in the measurement of piston
pipets with volumes from 1 μL to
50 μL. PTB Mitteilungen 1995;
- Lochner KH, Ballweg T, Fahrenkrog
HH. Investigations for the accuracy of
piston pipets with air cushion. J Lab
Med 1996; 20:430–40.
Dr. Lochner is Senior Research Scientist,
Fraunhofer-Institute for Silicate Research,
Bronnbach Branch, Bronnbach 28, 97877 Wertheim, Germany;
tel.: +49 9342 9221 701; fax: +49 9342 9221 799; e-mail: firstname.lastname@example.org. The author is indebted to Dr. Burkhard
Winter and Mr. Wolfhard Gögge of the German DIN Standards
Committee for Laboratory Instruments for their valuable input,
and Mr. Wilfried Langner and Mr. Jörg Barankewitz of Sartorius
AG (Göttingen, Germany) for their support and helpful suggestions
during the development of the SpeedCal semiautomatic gravimetric