Extreme Pipetting III: Death Valley Data
Extreme pipetting mission #3:
Editor'S Note: This is the third in a series of articles about pipetting, focusing on the impact of environmental conditions on data integrity. Part 2, which explored the effects of fluid temperature on data integrity, appeared in the September issue of PFQ.
As the ARTEL extreme pipetting expedition team set out for mission number three, only one thing was certain: it was about to get much hotter. Heading to Death Valley National Park in the desert spanning Nevada and California, the expedition'S goal was to test the effects of dry heat on pipetted volumes. As the sun beat down and the temperature rose, the cold temperatures at Mount Washington (site of mission number one) and the mild climate at Yellowstone National Park (where mission number two unfolded) seemed like distant memories to the expedition team.
A 3.3-million-acre desert, Death Valley is the hottest and driest area in North America. Its depth and narrow shape, as well as the four major mountain ranges separating it from the ocean, cause extreme desert conditions. Low elevations, lack of moisture, and an average of 300 days of sunshine result in an annual evaporation rate of 150 inches. Due to the rain-shadow effect of the mountains surrounding the valley, fewer than two inches of rain fall annually.
Luckily, laboratory technicians are not commonly subjected to conditions as extreme as those found in Death Valley. Laboratory environments are, however, often drier and hotter than ideal. Analytical instruments, ovens, incubators, freezers, and other devices using high power or open flames are common in laboratories and contribute heat and low humidity. Dry heat can also be caused by heating and air-conditioning systems. To ensure pipetting accuracy and precision and strengthen data integrity, laboratory scientists must understand and compensate for this source of error.
As the expedition team surveyed the landscape at Death Valley, humidity measured in at just 7% and temperatures soared to 44oC (111oF). It quickly became evident that setting up the pipette testing area in the shade was the wisest option.
The ARTEL PCS (Pipette Calibration System) would be used to verify the performance of common brands of laboratory air displacement pipettes in this dry, hot environment. Based on ratiometric photometry, the system is portable and unaffected by most environmental conditions. Due to the level of heat and aridity at Death Valley, however, the team took extra steps prior to testing the pipettes to verify that the PCS would operate accurately in the extreme conditions.
This was accomplished by using the PCS to measure liquid dispensed from a calibrated precision syringe. The PCS performed to specification, allowing the expedition team to isolate the hot, dry environmental conditions at Death Valley as the only possible source of pipetting error.
To test the performance of the air displacement pipettes, we collected 10 data points under four conditions from each pipette to account for any combination of the two variables of set volume and tip pre-wetting. Those conditions were: maximum volume and pre-wet; maximum volume and not pre-wet; minimum volume and prewet; and minimum volume and not pre-wet.
The first variable was volume. We tested each pipette at or close to its maximum and minimum volumes to identify which setting would be more prone to error. In addition, because many laboratories use pre-wetting protocols to offset liquid handling error, the team tested pipette accuracy and precision with and without pre-wetting. To test pipettes without pre-wet-ting, we changed pipette tips after each dispense, a protocol many laboratories use to avoid contamination. When pre-wetting, the team employed a rigorous procedure, including five aspirate/dispense cycles, prior to measuring the dispensed volume. The PCS automatically compared the dispensed volumes with the target volumes and quantified the resulting error.
The expedition team found pipettes significantly under-delivered in the dry heat of Death Valley. While the team partially reduced delivery errors by following the pre-wetting protocol, under-delivery persisted, and the pipettes were found to operate out of specification in most instances. As in previous missions, the pipettes at Death Valley exhibited greater errors when set to their minimum volumes than when set to their maximum volumes. Smaller-volume pipettes under-delivered by greater percentages than larger-volume pipettes.
For example, a 2 �L pipette set to deliver its maximum volume of 2 �L without pre-wetting under-delivered by 7%. Pre-wetting the tip used with this pipette prior to dispensing its maximum volume reduced the error from 7% to 4.8% (see Figure 1, above). The total inaccuracy, even with pre-wetting, was still outside the pipette'S maximum-volume specification of 1.5%, as set forth by the manufacturer (see Table 1, right).
That same 2 �L pipette, when set to deliver its minimum volume of 0.2 �L without pre-wetting, under-delivered by 34.7%. Pre-wetting reduced the error from 34.7% to 30.9%, but the pipette was still operating outside its minimum-volume accuracy specification of 12% (see Table 1). Again, pre-wetting mitigated, but did not fully compensate for, the volume delivery error caused by dry heat.
When working with larger liquid volumes, the hot and dry conditions still induced errors, but on a smaller magnitude. Without pre-wetting, a 20 �L pipette at its minimum volume under-delivered the sample by 16.8% (see Figure 2, p. 45), which is a significantly lower inaccuracy than exhibited by a non-pre-wet 2 �L pipette at its minimum volume (34.7% error in Figure 1). Pre-wetting the 20 �L pipette prior to dispensing its minimum volume of 2 �L reduced error from 16.8% to 7.4%. Here, pre-wetting reduced the error enough to allow the pipette to operate just within its minimum-volume accuracy specification of 7.5%.
When operating at its maximum volume, the 20 �L pipette, like the 2 �L pipette, exhibited smaller volume delivery inaccuracies than when operating at its minimum volume, totaling 5.5% without pre-wetting and 1.4% with pre-wetting (see Figure 2). The team'S studies in Death Valley also showed that error caused by dry heat was relatively consistent across pipette brands (see Figure 3, p. 45).
Evaporation Reduces Volumes
Pipetting error in the hot, dry environment of Death Valley occurs largely due to evaporation. In dry heat, evaporation of the sample fluid occurs within a pipette tip during the aspiration process. This evaporation increases the total volume of the gas phase, thereby increasing the air cushion in the pipette barrel. This increased air cushion prevents the pipette from aspirating the full, desired target volume, and less liquid is dispensed.
Volume is significantly affected because when 1 �L of liquid evaporates, it converts into more than 1,000 �L of gas, expanding by a factor of 1,250 to 1,450 depending on temperature. The evaporation of a minuscule amount of liquid inside the pipette tip can, therefore, have a large effect on pipetted volumes, especially when target volumes are in the microliter range.
Although laboratories do not commonly operate in environments as extreme as Death Valley, with 7% humidity and 44oC temperatures, laboratory conditions may lead to humidity as low as 15%. Humidity and temperatures within a laboratory can change throughout the course of a year, vary significantly between individual laboratories within the same building, and even differ in various sections of the same laboratory. It is also important to account for variations in temperature and humidity when quality control, research, and manufacturing projects are outsourced to contracting laboratories or transferred to another building or location within the company. To ensure data integrity and comparability across all laboratories, it is important to account for the effects of environmental conditions on pipetted volumes.
Ideally, laboratories would purchase systems that control humidity and temperature, but this could be costly. Alternatively, laboratory technicians can consistently monitor the humidity and heat within their facilities, determine the potential for evaporation, and adjust pipettes accordingly.
Another potential solution involves coordinating pipette calibration frequency with humidity cycles. Many laboratories calibrate their pipettes once or twice each year. Humidity often changes with the seasons or with other laboratory events, however. The magnitude of error recorded at Death Valley shows that it may be best to calibrate pipettes in accordance with humidity cycles. Considering the potentially widely varying temperature and humidity between laboratories within the same facility, the presented data underscore the importance of calibrating all pipettes under the same laboratory conditions in which they are used. This will account for any errors caused by the laboratory environment.
Considering the potentially widely varying levels of temperature and humidity between laboratories within the same facility, the presented data underscore the importance of calibrating all pipettes under the same laboratory conditions in which they are used.
Lastly, the expedition team strongly recommends that laboratory technicians pre-wet pipette tips prior to use. While pre-wetting did not fully compensate for pipetting inaccuracy under the extreme conditions of Death Valley, it consistently reduced the magnitude of error.
What'S Next?The extreme pipetting expedition team has provided scientific insight for laboratories on how barometric pressure, thermal disequilibrium, and dry heat affect pipetted volumes and data integrity. Look for the February/March 2008 issue of Pharmaceutical Formulation and Quality, which will discuss the findings of mission #4 of the expedition, testing the effects of humidity on pipetted volumes. The location for mission #4 will be announced shortly.