Beginner’s guide

to automated liquid handling

Anatomy of automated liquid transfers

This is just some of the theory of how fluid dynamics are affected by pipetting conditions and how this influences the pipetting technique. This guide also covers some basic liquid handling language and offers advice on mitigating against commonplace errors. This article is not an exhaustive guide; the best learning comes from hands-on practice, learning from mistakes, and continuous improvement.

Pipetting techniques

Forward pipetting

Also known as direct pipetting. The technique most commonly used with a manual pipette.
The aspirate is equal to the dispense volume. This technique is more likely to result in under-delivery of volume. Should be used in combination with a blow out and an air gap to be most effective.

Reverse pipetting

The aspirate is greater than the dispense volume. This technique is more likely to result in over-delivery of volume. This is best for both highly viscous and highly volatile liquids as well as low volume pipetting. This is because it ensures the minimum target volume is reached.

The effects of liquid temperature on pipetting volume accuracy

Lower temperatures increase viscosity and surface tension of liquids (maybe not for ethanol!); the opposite is true for hot liquids. Thus the speed at which a cold liquid flows in and out of the pipette is slower than for a hot liquid. 

The difference between the ambient temperature and the liquid temperature also has an effect. When the aspirated liquid is cooler than the ambient air, the dead air volume will be cooled on contact. The temperature drop causes the dead air pressure to increase and the air volume to shrink. As such, a slightly higher volume of liquid is drawn into the pipette, resulting in a larger aspirate volume. If the liquid is hotter than the room then the opposite occurs, and the aspirated volume might be smaller. In general, cold liquids over-deliver, where hot liquids under-deliver. The greater the difference in temperature between the liquid and the ambient environment, the greater the degree of error.

Ways to mitigate the effects of temperature: 

• Minimise transfer at extreme temperatures
• Allow the liquids to come to room temperature before transfer (if they aren’t temperature-sensitive)
• Pre-wet step to equilibrate the tip conditions to the liquid 
• Reduce the dead air volume

The effects of your lab environment

A pipette calibration lab will regulate its environment to be 20 °C and its humidity to be greater than 50%, all to minimise evaporation and optimise pipette accuracy. Humidity and temperature both determine evaporation rates and liquid viscosity. Since most pipettes rely on creating a pressure differential to move liquid, even the altitude of the lab affects pipetting. The pressure differential will be weaker at Kathmandu than it would in Sydney harbour and wouldn’t aspirate as great a volume. 

This affects how you pipette in your lab, but you subconsciously account for this whenever your experiment is adjusted to improve accuracy. However, the same workflow could be run using the same robot but in two different labs and the results might differ.

The effects of dead air space

The pressure in the dead air space will always equal the pressure (and other conditions) of the lab because it’s sharing the same air space. When the pipette aspirates liquid, the dead air space becomes contained and its conditions will be influenced by the liquid volume. 

Even if the liquid and the lab temperature are equal, there will still be molecules evaporating and condensing in the dead air space. If evaporation is greater than condensation then the vapour pressure (pressure that the gaseous molecule exerts on the liquid molecule) will increase. The dead air space will try to equalise with the environmental pressure so the space may expand slightly forcing the liquid volume towards the pipette tip bore (exit). This could create drips, reducing volume accuracy in your experiment.

The degree to which this could affect your data will vary depending on the application. Low humidity and atmospheric pressure in the lab, low boiling point and high liquid temperature all increase vapour pressure, which would increase the error.  

Ways to mitigate the effects of dead air space: 

• Use a travelling air gap to create an air space buffer beneath the liquid sample to prevent drips
• Pre-wet step to equilibrate the tip conditions to the liquid 
• Utilise reverse pipetting and make the aspiration greater than the dispense volume to guards against dispense accuracy errors if some of the volume drips

Fiona Kemm MRes | Scientist

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Fiona is a vital member of our Research team, rigorously testing our robots to ensure scientists don’t break them. With no prior robotics experience, she was the ideal guinea pig for our world-class user experience and support. Holding a BSc in Biochemistry and an MRes in Molecular Microbiology, Fiona brings extensive hands-on expertise she applies across departments, supporting both users and internal teams. From writing insightful web articles to specialising in SQWERTY, Fiona ensures our innovations perform flawlessly, helping customers focus on the creative and interpretive aspects of science that can’t be automated.