RESULTS
MISSION 2 – YELLOWSTONE NATIONAL PARK
The Effects of Pipetting Warm and Cold Fluids on Pipette Performance
The volumes of aqueous samples, which were not in thermal equilibrium with the
pipettes and tips used to deliver them, exhibit significant deviation compared
to volumes pipetted using thermally equilibrated samples, pipettes and tips.
The results obtained for delivering samples, which were in ambient temperature
equilibrium with the tips and pipettes used for delivery, served as reference
volumes to which the results obtained for warm or cold samples were compared.
Pipetting cold samples (1 – 4°C) resulted in over-delivery of up to 37%, while warm liquids (45°C) were under-delivered by up to 23%. Pipetting errors increased as the set
volume of the pipette decreased. When pipettes were used at their minimum
specified volume range, significantly larger errors were observed as compared
to using them at their nominal volume setting.
Figure 1 shows the errors induced by pipetting warm and cold liquids with a 2 μL pipette, set at its nominal volume (right graph) and at 10% of its nominal
volume (0.2 μL, left graph).
Figure 2 summarizes the results obtained for a 20 μL (nominal) pipette. It is obvious that the induced error is larger when the
pipette is used at its smallest volume setting, as compared to its nominal set
volume. The observed errors are smaller for this larger volume pipette compared
to the observed errors for the tested 2 μL pipette (Figure 1).
Figure 1. Pipetting of warm and cold samples with a 2 μL pipette, set at 0.2 μL (left graph), and at 2.0 μL (right graph).
Figure 2. Pipetting of cold samples with a 20 μL pipette, shows a larger error when set at 2.0 μL (left graph) than at its nominal volume of 20.0 μL (right graph). The magnitude of the induced error is smaller than for the 2 μL pipette (Figure 1).
Extreme weather conditions encountered in Yellowstone National Park during this
mission of the Extreme Pipetting Expedition made it impossible to complete data
collection of data points for all pipettes at all temperatures.
The significance of this field data, collected in an environment emblematic for
real-life laboratory conditions, warranted proper scientific investigation of
the errors resulting from pipetting liquids which are warmer or colder than the
used pipettes and tips.
The evaluation of three commonly used variable volume pipettes (20 μL, 200 μL, and 1000 μL) from three leading manufacturers is shown in Figures 3, 4, and 5. The same test protocol as in the field was followed, but under carefully
controlled laboratory conditions. As pipetting heated samples at 60°C is not uncommon in many procedures, pipette testing at this temperature point
was added to the protocol.
It is clearly visible in Figure 3 that all of the pipettes, at each tested volume setting, over-delivered cold
samples, which were thermostatted at 4°C.
Warm samples, in contrast, were consistently under-delivered as shown in Figure 4 (samples at 37°C) and Figure 5 (samples at 60°C).
All of the above results also indicate that the error associated with pipetting
warm or cold liquids is significantly larger for pipettes designed to deliver
small volumes. It is furthermore obvious, that errors increase when the
pipettes were used at or close to their minimum specified volume ranges, as
compared to their nominal volumes.
These laboratory results are in good agreement with the field results from
Yellowstone National Park.
THE CONCLUSION
Researchers who are pipetting warm or cold liquids need to be aware that this
technique is prone to introduce significant errors into common laboratory
procedures. Cold liquids tend to be delivered in excess quantity, while warm
liquids tend to be under-delivered. Depending on pipette manufacturer, volume
set point and temperature of the sample, these errors can exceed 65%, with
small volumes to be impacted most.
Whenever possible, it is recommended to transfer liquids that are equilibrated
to room temperature. When using protocols necessitating the handling of cold or
warm liquids with an air displacement pipette, it is recommended that the
researcher determines the pipette inaccuracy of the used
pipette/tip/temperature combination prior to the experiment. Since it is not
always feasible to determine the precise aberration from the calibrated volume
at any given temperature, volume, and tip combination, everyone interpreting
the data should be aware of the potentially very significant error introduced
by pipetting warm and cold liquids.
Figure 3. Pipetting liquids thermostatted at 4°C with pipettes set at or close to the specified minimum and nominal volumes,
results in over-delivery of the samples.
Figure 4. Under-delivery of samples thermostatted at 37°C.
Figure 5. Errors associated with pipetting warm samples increase with increasing sample
temperature, as shown here for 60°C warm samples.