Pause & Effect: How timing is critical for good pipetting technique

pipetting-techniqueIn a recent blog post about pipetting technique, Is your pipetting technique causing problems? I talked about the importance of prewetting the pipette tip for the most accurate pipetting.

Today I’d like to focus on another common mistake I see people making during pipette training sessions—pipetting too fast.

Pace yourself

Yes, you heard that right, you can actually affect both the accuracy and precision of your pipetting by pipetting too quickly, too slowly, or not pausing for the right amount of time after aspiration. For the most part, the problem I see during pipette training sessions is pace:  people pipetting too quickly and not pausing long enough after aspirating.  Usually I witness pauses that last somewhere between 0.25 – 0.33 seconds, whereas studies have found 1 second to be optimal (Figure 1). We all have so much to do that in an effort to be efficient, many of us end up rushing our pipetting without realizing the consequences—higher relative inaccuracy (RI) and higher coefficients of variation (CV), which ultimately lead to lower quality data. In fact, after discussing this “pause and effect” at a recent training session, the customer realized that this issue is so critical, she requested that we specifically talk to her group about “slowing down!”

What’s behind the “speed trap”?

The issue is movement of liquid into the pipette tip. When you pipette, you first depress the plunger, immerse the tip in the sample, and then release the plunger which causes liquid to enter the tip as the plunger moves back to its original position. But movement of the liquid into the tip is not instantaneous—there’s surface tension that needs to be overcome before the liquid enters the tip. In addition, friction between the liquid and the tip can slow down upward movement of the liquid. What this means is that once the plunger returns to its original position, the liquid usually hasn’t finished moving into the tip. Therefore, if you remove the pipette tip from the liquid too quickly before dispensing, you can end up transferring less volume than you intended.

We did a study on pause time after aspiration and found an optimum at 1 second (Figure 1). Interestingly, you end up with less volume both if you don’t pause long enough (0 seconds), and if you pause too long (5 seconds).  We suspect that the problem when you pause for too long is that evaporation of liquid inside the tip causes an expansion of the air volume, which slowly pushes the liquid back out of the tip.


Figure 1

Similar forces are likely to play a role in inaccuracies and imprecisions when the rate at which you release and depress the plunger during aspirates or dispenses—is not smooth, regular, or at the right speed.

So, pay attention to the little details of pipetting, such as speed and the pause before removing your tip from the liquid, for better data quality.

Learn more about good pipetting technique in Artel’s Best Practices for the Use of Micropipets.

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.