Good pipetting technique—don’t go in too deep (or too shallow)
Did you know that an ASTM standard specifies how far you should immerse a pipette tip?
One of the things I do at Artel is visit customer labs and conduct pipette proficiency certification seminars. These classes can be pretty surprising for analysts and scientists alike, some of whom may have been pipetting for a decade or more but find out that their pipetting technique can still be improved. From minor adjustments to hand holding—literally, I’ve had to physically move the hand and fingers of one twenty-year pipetting veteran to improve his technique—I’ve observed a wide range of pipetting challenges. But in my experience, there are three specific techniques that frequently lead to variability:
1. Not pre-wetting the pipette tip (read Digest post)
2. Not pausing long enough at the top of the stroke when aspirating (read Digest post)
3. Not immersing the pipette tip deep enough or immersing it too deep
In this Digest post, I’d like bring your attention to tip immersion depth, which is so important to accuracy and precision that an ASTM standard says this about it:
[7.2.2.4] Immerse the pipet or pipet tip into the liquid to be pipetted to, and maintain it at the following depth: 1
Volume, μL | Recommended Immersion Depth, mm | ||
1 to 100 | 2 to 3 | ||
101 to 1000 | 2 to 4 | ||
1.1 to 10 ml | 3 to 6 |
Tip immersion depth is one of those many little details that you need to think about when you pipette, in order to get the best performance. Like many of the other issues with pipetting technique, it has to do with the physics of pipetting.
This tip immersion is too shallow
The problems that occur when a pipette tip is immersed too shallowly are pretty easy to understand—if you don’t immerse your tip deeply enough, when you aspirate you run the risk of the tip ending up above the liquid before the plunger reaches the top of the stroke. The end result is air in the pipette tip and too little volume transferred.
This tip immersion is too deep
Sometimes a researcher will notice air in the pipette tip, realize that the tip had been too shallow, and will overcompensate by placing the tip in deeper than is optimal. One issue that can arise from this is actually touching the bottom of the tube, well, or vial you’re aspirating out of. This can partially block the opening of the tip, reducing the volume of liquid entering the tip. The end result is underdelivery of sample.
Even if your tip is not touching the bottom of the vessel, you can still run into problems if it’s too deep. Increased hydrostatic pressure can force extra liquid into the tip, leading to aspiration of too much volume. In addition, because more of the tip is immersed in the sample, the risk of carrying over droplets adhering to the outside of the tip also increases – the risk of carryover is proportional to the surface area in contact with the liquid. In this scenario, you’ll end up overdelivering sample.
Studies show that improper immersion can alter the delivered volume by 2.2%.2
This tip depth is just right
To get optimal accuracy and precision out of your pipette, remember to pay attention to how deep your pipette tip is immersed into your sample— 2–3 mm for pipette volumes of 1–100 μL, 2–4 mm for 101–1000 μL, and 3–6 mm for volumes larger than 1 mL. Your data will be better for it.
Find more tips for good pipetting technique in Artel’s 10 Tips to Improve Your Pipetting Technique.
Additional Resources
- Publication: Best Practices for the Use of Micropipets
- Lab Report 2: Impact of Pipetting Technique
- Video and Wall Poster: 10 tips to Improve Your Pipetting Technique
- Blog: Tips on How to Pipette
References
- ASTM E1154-14(2014). Standard specification for piston or plunger operated volumetric apparatus. ASTM International, West Conshohocken, PA;
- A.B. Carle, D. Rumery, and G. Rodrigues. Best Practices for the Use of Micropipets. American Laboratory. June/July 2014. 46(5):14-17.
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.
