Extreme Pipetting IV: It’s Not the Heat, It’s the…

By Rumery, D.; Carle, B. | Publication

Pipettes perform exceptionally well in high humidity

Fresh on the heels of Mission 3 at the stiflingly hot Death Valley National Park, the Artel Extreme Pipetting Expedition team looked forward to visiting the temperate and humid Olympic National Park. Known for its lush, rainforest-like conditions, Olympic is home to 266 glaciers, more than 60 miles of rugged Pacific coastline, and over 140 inches of rainfall each year. It also has the Northwest’s largest remaining acreage of undisturbed rainforests.

Pipetting-in-humidity

But the Extreme Pipetting Expedition did not travel cross-country from Portland, Maine to west of Seattle, Washington to sightsee. The team visited Olympic National Park to test pipette performance in high humidity for Mission 4 of 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 their 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 that it is difficult to separate temperature and humidity when analyzing pipette performance.

It is clear that 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 called 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 two equations can be used:

1. Evaporation Potential = Pw, saturated – Pw, ambient
2. Evaporation Potential = Pw, saturated x (1 – Relative Humidity)

The partial pressure of water in air (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. This is what occurs in most pipette calibration laboratories – temperature is held constant at 20 °C, and relative humidity is increased to decrease the evaporation rate and improve pipette performance.

The Experiment

To study pipette performance in a high humidity and low evaporation potential environment, Artel sought a damp, temperate location. Given that Olympic National Park is usually temperate and humid in the late summer and early fall, Artel chose this time period for Mission 4. Upon arriving at Olympic, the unseasonably cold temperatures were quickly apparent, with the ambient temperature about 6 °C cooler than the average normal temperature.

Undeterred, Artel set up to test pipette performance at Olympic’s Rialto Beach, on the north side of the Quileute river. At 14 °C and 74% relative humidity, because of the low temperature and high relative humidity, the site had a very low evaporation potential of just 4.5 millibars (mbar).

As in previous missions, pipette performance was tested in the field using the Artel PCS® Pipette Calibration System. Based on ratiometric photometry, the system is portable and unaffected by most environmental conditions. The PCS was used to measure volumes dispensed by a 2, 20, 200 and 1000 microliter pipette. The system automatically compared the actual dispensed volumes to the desired target volumes and quantified the resulting error.

Artel found that in the very humid environment at Rialto Beach, the pipettes performed extremely well. Figure 1 shows the tested pipettes dispensed accurately, with average inaccuracy of only -0.35%, with -1.55% the largest inaccuracy recorded.

Figure 1.  Inaccuracy (normalized) in Various Environments

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Figure 2 shows that 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.

Figure 2.  Imprecision in Various Environments

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After returning from Rialto Beach, Extreme Pipetting Expedition members noticed that at Olympic Inn, their accommodations for the Mission, relative humidity measured exactly 60% and the temperature 20 °C – the ideal regulatory specification for pipette calibration laboratories. After testing pipettes at the Olympic Inn, Artel found that the pipettes also performed well, as expected. Average inaccuracy was -0.11% and average imprecision ratio was 0.68.

The pipettes performed similarly at Olympic Inn and Artel’s controlled calibration laboratory because both locations had almost equal evaporation potential. The evaporation potential was 10 mbar in the calibration laboratory and 9.4 mbar at Olympic Inn.

Pipette Performance in Typical Laboratories

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

First, Expedition members tested pipettes in Lab A with drier conditions at 22% relative humidity and 21 °C. Pipettes were also tested in Lab B with more humid conditions at 40% relative humidity and 22 °C.

In Lab B, the more humid laboratory with an evaporation potential of 16 mbar, average inaccuracy was 0.05% and 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 average imprecision ratio was 7.10, both statistically significant errors. The data show that drier laboratories are more prone to liquid handling error. The data also show a statistically significant degradation in pipette performance in the 20% humidity environment versus the 60% humidity environment.

The data are plotted on Figures 1 and 2, which indicate that the greater the evaporation potential, the more pronounced are pipetting inaccuracy and imprecision.

What Does this Mean?

As was found during Mission 3 of the Extreme Pipetting Expedition at Death Valley, 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 being pipetted, the pipette tip becomes humidified, causing the delivery volume to increase with each dispense. This trending from small volumes closer to the target volume causes imprecision and variability in data.

Conversely, in humid and cooler environments, 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. Pipetting becomes a more stable, repeatable process.

Laboratories with several conditions may be prone to pipetting error caused by the absence of humidity. First, laboratories with dry and warm environments may experience under-delivery during pipetting. In addition, laboratories with humidity levels that fluctuate throughout the year may also experience variability in pipetting. Lastly, 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.

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 discussed following the release of the Death Valley results, Artel strongly recommends that laboratory technicians pre-wet pipette tips prior to use. Pre-wetting the tips will humidify pipettes before dispensing to improve overall performance, reduce the risk of evaporation, and result in more consistent volume dispenses.