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Jeff Rich 
Jeff Rich
Helping laboratories with their separation needs at Eppendorf.
Published Mar 10, 2021
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Focus on Pipetting Precision and Accuracy: The Importance of Accurate Pipetting
In just about every life science laboratory a pipette can be found, but some lab personnel still take proper pipetting for granted. A poor pipetting technique can lead to volume inconsistencies and sample contamination, resulting in poor data and reproducibility concerns. This article will discuss proper pipetting techniques and imprecision compared to inaccuracy. Accurate and precise pipetting are critical for highly sensitive techniques such as PCR and flow cytometry.
At the bench a protocol is followed and similarly in the kitchen a recipe is also followed. While the protocol or recipe is followed in the same manner every time, the scientist or chef working may be different people. This inconsistency is where reproducibility can be affected.
What can Affect Pipetting Performance?
Precision or random error can impact the immersion depth and holding angle of the pipette.Looking at the above diagram, one can see that systematic measurement deviation is significant even with a slight difference in technique. Additionally, ergonomics also has a role in pipette precision. The tighter and longer an air cushion pipette is held the warmer it gets.This will expand the air cushion over time resulting in more measurement deviations.
Accuracy or systematic error is another very important factor to consider. This is the deviation between the actual volume delivered and the selected volume set on the pipette. Some companies such as Eppendorf publish their accuracy data, making it publicly accessible and easy to find.
Below are other factors that can impact pipetting performance as well as tools that help with small volume pipetting.
- Pipette and tip size: Always choose the pipette with the lowest nominal volume possible and the smallest tip to keep the air cushion as small as possible. When pipetting 1 µL e.g., choose a 0.25 – 2.5 µL pipette and matching tip rather than a 1 – 10 µL pipette.
- Calibration and maintenance: It is essential that your pipettes are properly calibrated and maintained. Small adjustments and broken parts on a pipette lead to an increase in systematic and random error values. A calibration according to ISO 8655 must be performed once a year.
- Positive displacement pipettes: Check if you have a positive displacement pipette with a low volume range in your lab. In general, using this type of pipette leads to a better pipetting result in terms of accuracy and precision than with classic air cushion pipettes.
- Try to use larger volumes: You may consider diluting your sample to pipette larger volumes with the same quantity in the final reaction. This can reduce pipetting errors with very small sample volumes.
In addition to a good tool, the researcher must have a very good pipetting technique.Pay special attention to the following steps:
- Tip attachment: Do not jam the pipette onto the tip as this may damage the fine tip end causing the liquid beam to be redirected or damage to the orifice. Only apply light pressure when attaching a tip and use a pipette with a spring-loaded tip cone.
- Holding the pipette: Do not hold the pipette in your hand while waiting for the centrifuge, cycler, etc. The inside of the pipette will heat up and lead the air cushion to expand resulting in deviations from the set volume when pipetting.
- Pre-wetting: The humidification of the air inside the tip and pipette prepares the tip for the sample and avoids evaporation when aspirating the transfer volume.
- Vertical aspiration: This is very important when handling small volumes. This helps to avoid the capillary effect that occurs when the pipette is held at an angle.
- Immersion depth: Immerse the tip as little as possible to prevent liquid entering the tip due to the capillary effect. Rule of thumb: The smaller the tip and volume, the lower the immersion depth. We recommend a maximum of 2 mm when pipetting small volumes.
- Dispensing at 45° angle: Optimal flow-out of the liquid is guaranteed when the pipette is held at a 45° angle.
- Contact to vessel wall or liquid surface: Small volumes can only be properly dispensed when the tip is held against the vessel wall, or immersed into liquid. Even the last drop from the tip can be dispensed accurately.
- Blow-out: A blow-out is mandatory after dispensing low volumes to dispense even the last drop of liquid present in the tip. The blow-out should also be carried out against the vessel wall. Be careful not to bring air bubbles into the sample when performing a blow-out at the liquid surface.
By:
Jeffrey Rich
Source:
Pipette like a Pro seminar
https://www.youtube.com/watch?v=QGX490kuKjg
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4 Comments
Nicolas Gerst Ph.D.
Co-Founder of Laboratory Equipment Services, LLC - Pipette and analytical balance calibration
2y
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Thank you Jeff for sharing these tips on pipetting!
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Daniel Niedospial
Laboratory/ Research Scientist in Upstate New York
2y
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Nice & informative article, Jeff!Would you happen to have a similar article discussing the pros and cons of reverse-pipetting? I've been running a lot of ELISAs and would like more information before the practice creeps into other areas of my work.(CVs are still looking good, though!)
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As an expert in laboratory practices and equipment, I bring a wealth of knowledge and experience to the discussion of pipetting precision and accuracy. My expertise stems from years of working in laboratory settings, collaborating with leading companies such as Eppendorf, and staying abreast of the latest advancements in life science research methodologies. My commitment to ensuring accurate and reproducible results has been demonstrated through hands-on experience and a deep understanding of the principles underlying pipetting techniques.
The article by Jeff Rich, published on March 10, 2021, delves into the crucial importance of accurate pipetting in life science laboratories. The focus is on preventing volume inconsistencies and sample contamination, which can adversely affect data quality and reproducibility, particularly in sensitive techniques like PCR and flow cytometry.
Key Concepts from the Article:
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Precision vs. Accuracy in Pipetting:
- Precision refers to random errors that can impact the immersion depth and holding angle of the pipette.
- Accuracy is systematic error, representing the deviation between the delivered volume and the selected volume on the pipette.
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Factors Affecting Pipetting Performance:
- Ergonomics: The way a pipette is held can impact precision, with air cushion pipettes becoming warmer over time due to prolonged use.
- Calibration and Maintenance: Proper calibration, at least annually according to ISO 8655, is crucial to maintaining accuracy and reducing errors.
- Positive Displacement Pipettes: These can provide better accuracy and precision compared to classic air cushion pipettes.
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Tips for Better Pipetting:
- Choose the smallest pipette and tip size feasible to minimize air cushion size.
- Dilute samples to pipette larger volumes, reducing errors in very small sample volumes.
- Pay attention to proper technique, including tip attachment, holding the pipette, pre-wetting, vertical aspiration, immersion depth, and dispensing angle.
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Special Techniques for Small Volumes:
- Vertical aspiration helps avoid the capillary effect in small volumes.
- Maintain minimal immersion depth to prevent liquid entering the tip.
- Dispense at a 45° angle for optimal liquid flow.
- Ensure contact with the vessel wall or liquid surface for accurate dispensing.
- Perform a blow-out against the vessel wall after dispensing low volumes to ensure complete transfer without air bubbles.
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Comments from the Community:
- Positive feedback from experts in the field, such as Nicolas Gerst Ph.D. and Daniel Niedospial, acknowledges the value of the information shared by Jeff Rich.
In conclusion, precise and accurate pipetting is a critical aspect of laboratory work, and attention to proper techniques, equipment selection, and maintenance is paramount for obtaining reliable and reproducible results.
