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.

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.
For more on how the PCS works, click here.

Methods

Experimental methods 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. are described within each mission. Because pipette user technique is so important, all operators on the Extreme Pipetting Expedition are trained in 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. The Extreme Pipetting Expedition can 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 can 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.