Pipettes perform exceptionally well in high humidity

In The Lab: PIPETTES

DOREEN RUMERY AND A. BJOERNCARLE, PHD

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ALL IMAGES COURTESY OF ARTEL

Extreme Pipetting IV: It's Not the Heat, It's the...

F resh from mission number three, completed in stiflingly hot Death Valley National Park, the ARTEL Extreme Pipetting Expedition team looked forward to visiting the temperate and humid Olympic National Park (Port Angeles, Wash.). Known for its lush, rain forest-like conditions, Olympic is home to 266 glaciers, encompasses more than 60 miles of rugged Pacific coastline, and enjoys over 140 inches of rainfall each year. It also has the Northwest's largest remaining acreage of undisturbed rain forest.

But the Extreme Pipetting Expedition had not traveled all the way across the country from Portland, Maine, to a national park west of Seattle just to sightsee. The team was visiting Olympic National Park to test pipette performance in high humidity-mission number four on its scientific journey.

Understanding how pipettes perform in high humidity is critical for laboratory data integrity. Humidity levels can vary across laboratories-and even within a laboratory itself. In addition, according to regulatory standards, pipette calibration laboratories must control relative humidity at greater than 50%, while working laboratories are often significantly drier. By visiting Olympic National Park, ARTEL sought to determine how pipettes behave in a humid environment and whether varying humidity levels affect pipetting accuracy and precision.

Defining Evaporation Potential

Before testing the effect of high humidity on pipetted volumes, ARTEL evaluated the relationship between humidity and temperature. A critical finding from Death Valley, where ARTEL measured dry heat's effect on pipetting, was the difficulty of separating temperature and humidity when analyzing pipette performance.

Figure 1. Inaccuracy (normalized) in various environments.

Pipetting in dry environments causes evaporation to occur within the pipette tip, leading to under-delivery of aqueous solutions. In an environment with constant relative humidity, greater evaporation occurs in a warm temperature environment than in a cold temperature environment. The rate of evaporation is proportional to a thermodynamic force-the evaporation potential-which is defined as the difference between the partial pressure of water in air (Pw) at saturation conditions (100% relative humidity) and the actual partial pressure of water in air at ambient conditions. Understanding a laboratory's evaporation potential is the first step in studying how humid conditions affect pipetted volumes.

To determine a location's evaporation potential, either of the following equations can be used:

• Evaporation potential = Pw, saturated - Pw, ambient; or

• Evaporation potential = Pw, saturated * (1 - relative humidity).

The Pw at saturation conditions is almost entirely dependent on temperature. As temperature increases, the amount of water that can be held in the air also increases. If relative humidity is held constant, raising the temperature increases the evaporation potential and the evaporation rate inside the pipette. On the other hand, if temperature is held constant, raising the relative humidity decreases the evaporation potential. In most pipette calibration laboratories, temperature is held constant at 20^oC and relative humidity is increased in order to decrease the evaporation rate and improve pipette performance.

The Experiment

To study pipette performance in an environment with high humidity and low evaporation potential, ARTEL needed a damp, temperate location. Because conditions in Olympic National Park are usually temperate and humid in the late summer and early fall, ARTEL chose this season for mission number four. Upon arriving at Olympic, the team immediately noted unseasonably cold temperatures, with the ambient temperature about 6^oC cooler than the average temperature at the same time of year.

Undeterred, ARTEL set up its testing tools at Olympic's Rialto Beach, on the north side of the Quillayute River. Because of the low temperature and high relative humidity (14^oC and 74% relative humidity), the site had a very low evaporation potential of just 4.5 millibars (mbar).

Figure 2. Imprecision in various environments (CV: imprecision results).

As in previous missions in the field, the team tested pipette performance using the ARTEL Pipette Calibration System (PCS). Based on ratiometric photometry, the system is portable and is not affected by most environmental conditions. The PCS was used to measure volumes dispensed by 2, 20, 200, and 1,000 �L pipettes, automatically comparing the actual dispensed volumes to the desired target volumes and quantifying the resulting error.

ARTEL found that the pipettes performed extremely well in the high humidity of Rialto Beach. Figure 1 (left) shows the tested pipettes dispensed accurately, with average inaccuracy of only -0.35%; -1.55% was the largest inaccuracy recorded. Figure 2 (below) shows the pipettes also performed consistently at Rialto Beach, with all but one of the pipettes recording imprecision results (CV) very close to the pipette specification.

After returning from Rialto Beach, Extreme Pipetting Expedition members noticed that at the Olympic Inn where they were staying, relative humidity measured exactly 60% and the temperature was 20^oC-the ideal regulatory specification for pipette calibration laboratories. As expected, pipettes tested at the Olympic Inn also performed well. Average inaccuracy was -0.11% and average imprecision ratio was 0.68.

The pipettes performed similarly at the Olympic Inn and at ARTEL's controlled calibration laboratory because the evaporation potential was almost equal at the two locations: 10 mbar in the calibration laboratory and 9.4 mbar at the Olympic Inn.

Performance in Typical Laboratories

While ARTEL seems to have found the perfect environment for accurate and precise pipette performance, laboratories are not typically as humid as Olympic National Park. Relative humidity of 15-40% is more common in laboratories. To determine if pipettes behave differently in typical laboratories than they do in humid laboratories, ARTEL tested pipettes in environments with varying degrees of evaporation potential.

When pipetting, the instrument is consistently humidified, resulting in little or no evaporation. Without evaporation, volume dispenses are more consistent and accurate and pipetting becomes a more stable, repeatable process.

First, expedition members tested pipettes in Lab A with 22% relative humidity at 21^oC. Pipettes were also tested in Lab B at 40% relative humidity and 22^oC. In Lab B-the more humid laboratory with an evaporation potential of 16 mbar-average inaccuracy was 0.05% and the average imprecision ratio was 0.73, indicating that pipettes performed well. In Lab A-with an evaporation potential of 20.6 mbar-average inaccuracy was -1.55% and the average imprecision ratio was 7.10, both statistically significant errors. The data show that drier laboratories are more prone to liquid handling error. A statistically significant degradation in pipette performance occurs in the environment with 20% humidity versus the one with 60% humidity.

The data plotted in Figures 1 and 2 indicate that the greater the evaporation potential, the more pronounced pipetting inaccuracy and imprecision. Complete data can be found at

What Does This Mean?

As the team found in Death Valley during mission number three of the Extreme Pipetting Expedition, dry heat causes pipettes to under-deliver aqueous solutions due to evaporation. When pipetting aqueous solutions, evaporation occurs within the pipette tip with the first aspiration, and less liquid is dispensed. Over time, as the pipette is continually exposed to the aqueous liquid, its tip becomes humidified, causing the delivery volume to increase with each dispense. This trending, from small volumes to volumes closer to the target number, causes imprecision and variability in data.

Conversely, in environments that are cooler and more humid, the evaporation potential is minimal. When pipetting, the instrument is consistently humidified, resulting in little to no evaporation. Without evaporation, volume dispenses are more consistent and accurate and pipetting becomes a more stable, repeatable process.

Several different types of laboratories may be prone to pipetting error caused by the absence of humidity. First, laboratories with warm, dry environments may experience under-delivery during pipetting. Laboratories with humidity levels that fluctuate throughout the year may also experience variability in pipetting. Finally, pipettes calibrated in pipette calibration facilities are more likely to operate out of specification when used in laboratories at higher temperature and lower relative humidity.

Fun Facts About Olympic National Park

• Ninety-five percent of Olympic National Park is designated wilderness by Congress.

• About 12 feet of rain falls each year on the west-facing valleys, sustaining the temperate rain forest.

• In 1977, a farmer digging a pond just outside Olympic National Park unearthed the remains of a mastodon, a huge elephant-like mammal that grazed ice age grasslands.

Several steps can be taken to reduce the risk of pipetting error caused by humidity changes. First, verifying the performance of liquid handling instruments in the environment in which they are used can help to eliminate humidity as a source of error. This practice will provide information about how pipettes perform in actual conditions and allow laboratories to adjust their pipetting processes accordingly.

Second, as ARTEL recommended following the release of the Death Valley results, laboratory technicians should wet pipette tips prior to use. Pre-wetting the tips to humidify pipettes before dispensing will improve overall performance, reduce the risk of evaporation, and result in more consistent volume dispenses.

On the Horizon

ARTEL has visited a number of extreme locations to provide greater insight into the effects environmental conditions can have on laboratory data integrity. During the Extreme Pipetting Expedition, the team conducted research on how extreme levels of barometric pressure, thermal disequilibrium, dry heat, and humidity affect pipetted volumes.

In response to feedback from concerned scientists and laboratory technicians, ARTEL plans to visit actual laboratories in which environment is believed to have an impact on data. ARTEL will test pipette performance under real-world working conditions-including dynamic effects such as temperature changes-to continue offering laboratories concrete guidance on how to optimize liquid handling operations.