Improving data quality and reproducibility (Part 2): keeping an eye on humidity

In this, the second of our four-part series, we visited Olympic National Park in Washington State, home to the Pacific Northwest’s largest remaining acreage of undisturbed rainforest to test how humidity affects pipetting.

Why humidity?

You might not think that humidity can affect the accuracy, precision and trueness of your pipetting, but the effects can be noticeable (Figures 1 and 2). Even if you keep your samples covered, the humidity and temperature of your lab can lead to under- or over-delivery of sample and a large coefficient of variation (CV). This is for two reasons—first, the better calibration labs (the ones you want to use) will calibrate your pipettes under specified, controlled conditions (20°C and 60% relative humidity) which are optimal for pipette performance. However, many research labs are not humidity-controlled and operate under ambient conditions, often between 15 – 40% relative humidity. Because of this discrepancy between labs, you might not get the same level of pipette performance in your research lab as certified by the calibration lab.

Figure 1.  Inaccuracy (normalized) in Various Environments

Pipetting inaccuracy at various levels of humidity. Go to the Extreme Pipetting Expedition article on Olympic National Park for the full experimental details.

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Figure 2.  Imprecision in Various Environments

Pipetting imprecision at various levels of humidity. Go to the Extreme Pipetting Expedition article on Olympic National Park for the full experimental details.

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The second reason humidity and temperature can affect pipette performance is that, together, they affect the evaporation rate of your sample, not just when it’s in the tube but also once inside the pipette tip. It is evaporation into the air cushion within your pipette tip that can really change your liquid handling performance. Let’s take a closer look at these factors.

The microenvironment inside the pipette tip impacts pipetting performance

Most handheld pipettes use air displacement to move liquid around. Once the tip is immersed into liquid, a cushion of air is trapped above it, forming a microenvironment within the pipette tip. This air cushion—also called the captive air volume or dead air volume—now interacts with the trapped liquid as described by the Ideal Gas Law:


which shows that the volume of the air cushion is affected by the number of molecules in the gas phase (n). If the relative humidity of the air cushion has not reached equilibrium with the liquid, microevaporation of the sample into the air cushion will happen, increasing the amount of gas, and reducing the amount of liquid that is aspirated and dispensed.

Note that pre-wetting the pipette tip can improve performance by increasing the relative humidity inside the pipette tip, but no amount pre-wetting can completely humidify a tip to 100% relative humidity.  Consequently, the temperature and humidity of the research lab compared to the calibration lab can still affect liquid handling performance.

Relative humidity and temperature affect evaporation rate

You can get more details on how humidity and temperature affect evaporation in the full Extreme Pipetting Expedition article on Olympic National Park, but briefly it’s as follows: The evaporation rate depends on the evaporation potential of a sample, which is defined as the difference between the partial pressure of water in air at saturation conditions (Pw,sat) and the actual partial pressure of water in air at ambient conditions (Pw). Because Pw,sat increases with temperature, the evaporation potential of a sample will depend on both temperature and humidity. If temperature is held constant, increasing relative humidity reduces evaporation potential and the evaporation rate inside the pipette tip, leading to more consistent pipetting. If relative humidity remains constant and temperature increases, the evaporation potential and the evaporation rate inside the pipette tip increases, leading to less consistent pipetting and under-delivery of sample.

A few last comments

What we illustrate here and in more detail in all of the Extreme Pipetting Expedition articles is how environmental factors can have a noticeable impact on your liquid handling performance, and thus the quality and reproducibility of your data. The larger take-home message is that while each of these individual impacts may seem to be so small that they’re negligible, taken together they add up. So for really good results that you can repeatedly trust, it’s worth it to pay attention to and optimize environmental impacts on pipetting.

Additional Resources

About the Author

Doreen Rumery

Doreen Rumery, Laboratory Technical Manager and Quality Control Manager at Artel, is a certified Medical Laboratory Technologist and ASQ Certified Manager of Quality/Organizational Excellence with more than 30 years in the clinical laboratory and manufacturing industries. Doreen oversees all laboratory activities at Artel, including technical operations, method validation, technician training and conformance with current regulatory standards.