Is your pipetting technique causing problems?

Jul 21, 2015

impact-of-pipetting-techniqueHandheld pipettes are an incredibly widespread tool in the life sciences, yet I’ve seen many scientists who are surprised to discover that they’re not pipetting as accurately or precisely as possible due to poor pipetting technique.  Seemingly small technique issues like tip immersion depth,  pause length, or speed of aspiration can all cause pipetting errors, but the most common mistake I see people making is failing to pre-wet the pipette tip.

During one training session, a customer showed amazingly consistent pipetting technique with her pipette set at 20 uL.  She used a new tip for each pipetted sample, and while consistent, the delivered volume was usually 0.5 µl below the 20 uL target volume – because she wasn’t pre-wetting the tip prior to pipetting.  This lack of accuracy, but good reproducibility, meant that if she re-ran an experiment, the results would be similar to the first run.  However, the lack of a laboratory rule on pre-wetting pipette tips (or having some scientists pre-wet and others not) could contribute to scientist-to-scientist variations, affecting overall reproducibility and the quality results her laboratory was striving for.

It may sound inefficient, but if you want to increase the accuracy and precision of your pipetting, you really need to make sure that the air cushion inside the tip and directly above the sample is properly humidified by performing several aspirate/dispense cycles.

When pipetting, microenvironments matter

Let’s take a closer look at what happens inside the pipette upon aspiration:

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 behaves according to the Ideal Gas Law:

PV= nRT

which shows that the volume of the air cushion is not only affected by temperature but also by the partial pressures of air and the liquid’s gas phase. These two gases strive to establish an equilibrium, which means that some of the sample liquid in the tip evaporates into the captive air volume. This evaporation has a dual effect:  Not only does it result in the loss of sample liquid, but also in the increase of the captive air volume, which in turn leads to the aspiration of less liquid volume.  Once the captive air volume is nearly saturated with the sample liquid’s vapor, this effect becomes increasingly negligible.

Pre-wetting your pipette tip reduces evaporation and thereby increases the volume accuracy of the dispensed liquid. In Best Practices for the Use of Micropipets, you can see a nice graph that shows some data on how accuracy is affected.

Proper pre-wetting technique

Studies on pre-wetting methods (see Lab Report 2: Impact of Pipetting Technique), lead to the following recommendation:

1. Set your pipette to the desired volume.
2. Immerse the tip into the sample and aspirate (learn more about the proper immersion depth in 10 Tips to Improve Your Pipetting Technique)
3. Pause for one second and then remove the tip from the solution.
4. Dispense back into the receptacle.
5. Repeat five times (per ISO recommendation).

Clearly, this procedure ends up taking time, but if accuracy and precision are critical, it’s worth it.  Alternatively, you can compromise so long as you are prepared for decreased accuracy and precision; for instance by performing only one round of pre-wetting. Just make sure everyone in your lab is using the same technique.

Beyond pre-wetting

While pre-wetting is among the most common issues affecting pipetting accuracy and precision, there are a number of other techniques you can employ to improve your pipetting. Learn more in Lab Report 2: Impact of Pipetting Technique, and in 10 Tips to Improve your Pipetting Technique and in the Best Practices article already mentioned. With knowledge and with focus on technique, you’ll be well on your way to becoming a much more accurate and precise pipette user.


Additional Resources


About the Expert

Candie Gilman

Candie Gilman, Training Product Manager for Artel Pipette Quality Management and Technique Certification programs, has coached hundreds of laboratory professionals, helping pipette users and quality assurance managers standardize their pipetting technique. In her hands-on seminars, participants gain measurably improved pipetting skills, as well as a new appreciation for ergonomics, pipette maintenance and industry and regulatory standards.