THE SCIENCE
HOW THE ENVIRONMENT AFFECTS PIPETTES
Nearly all handheld pipettes contain an internal volume of captive air that acts
as a coupling between the moving piston inside the pipette and the liquid that
is being aspirated and dispensed from the pipette tip. Like all gases, this
so-called “dead air volume” is springy: when pulling air into the tip, this gas expands slightly and then
compresses slightly as liquid is expelled. This springy coupling between the
piston and liquid creates an offset from ideal performance, which is corrected
during the calibration process. The magnitude of this correction depends on the
properties of the air, the tip and the liquid being pipetted - and those
properties vary depending on the environmental conditions.
The most important environmental conditions that need to be considered when
working with pipettes are temperature, barometric pressure and relative
humidity. For pipette calibrations these three conditions should be routinely
controlled and reported within the pipette calibration certificate. In fact, to
meet international pipette standards established in 2002 (ISO 8655 Part 2),
pipettes are required to be calibrated to deliver accurately under the
following environmental conditions.
Temperature = 20°C
Relative Humidity = 50%
Barometric Pressure = 101 kPa
It is important to note that it is rare to find a normal working laboratory that
precisely adheres to the environmental conditions specified in ISO 8655. So the
standard environmental conditions for pipette calibrations define a starting
point, but do not answer the need to know what happens in the real world
laboratory when the pipette is used under conditions that differ from
calibration in a controlled environment. Answering this question and drawing
attention to the implications for laboratory quality is the purpose of the
ARTEL Extreme Pipetting Expedition.
Some of these effects can be modeled or studied in the laboratory environmental
chamber. From the ideal gas law for example, we know that the air coupling
inside the pipette will become softer (less stiff) when barometric pressure
falls. When this happens the pipetted volume should decrease, at least in
principle. (Measuring this effect is the subject of the first mission in the
Extreme Pipetting Expedition – a trip to the Mount Washington Observatory.)
There is a distinct advantage in complementing the theoretical modeling (or
laboratory experiments) with actual Expedition data gathered in real world
environmental extremes. Both theoretical modeling and laboratory experiments
rely on the scientist having anticipated all of the complex and interdependent
environmental factors that might impact pipetting. Expedition data however, has
the advantage of including influences from all sorts of unanticipated
environmental factors that might come into play in a harsh and uncontrolled
environment.
Thus, the Expedition data will serve as a real world check on the validity of
theoretical models of pipetting performance and challenge ARTEL scientists to
go beyond the bounds of normal laboratory environmental thought. If the
Expedition data turns out to be in good agreement with theory, it will give us
confidence that all significant factors have been properly considered in the
theoretical model. On the other hand, disagreements may point to a need for
consideration
of unanticipated factors.
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Equipment
For the Extreme Pipetting Expedition Missions, adjustable volume pipettes from
three different popular manufacturers are included. For each manufacturer, four
different volume sizes are available including 1000, 200, 20 and 2 microliter
sizes. The manufacturer’s recommended pipette tips are used for each pipette.
The measurement system used in the Extreme Pipetting Expedition is the ARTEL PCS
- a portable photometric volume measurement system that is used in thousands of
laboratories worldwide. In contrast to other common means of pipette
calibration, the ARTEL PCS is reliable, accurate and precise in even the most
demanding environments making it the ideal volume measurement tool for the
Extreme Pipetting Expedition.
Methods
Experimental methods will vary slightly from one part of the Expedition to the
next because each independent mission is designed to test a different
environmental influence. Specific details of the test protocol, such as the
precise order of testing, number of data point replicates, etc. will be
described within each mission. Because pipette user technique is so important,
all operators on the Extreme Pipetting Expedition are trained using the ARTEL
Method, and have been tested and synchronized with one another to control for
operator-to-operator variation. When a base case is needed for interpretation
of the field date, pipette performance is compared to results obtained in ARTEL’s Accredited (ISO 17025) pipette calibration laboratory in Westbrook, Maine.
Summary
Environmental factors can have a significant effect on the accuracy and
precision of liquid delivery. It is anticipated that the Extreme Pipetting
Expedition will help to advance the state of knowledge of this influence and
also serve to help laboratories become more aware and informed about this
source of bias and imprecision in liquid measurement. The lessons learned from
the Expedition are expected to assist scientists to be better prepared to
anticipate this error, estimate or measure its impact, and take corrective
actions to improve the quality of data generated in every laboratory where
pipettes are used.
