The pipette is a reliable precision instrument that has been used and trusted for many years. However, as with many forms of instrumentation, a pipette will perform only as well as the operator’s technique allows.
Differences in technique – some more than others – can alter delivery volumes. With increasing demand for accuracy, quality and productivity the importance of understanding and developing optimal pipetting technique becomes imperative. Let’s review the results of a study conducted at Artel to determine these differences and assess their impact upon pipetting accuracy.
The reference pipetting method used in each of these experiments was as follows: The pipette mechanism was “warmed up” by gently depressing and releasing the plunger 15 to 20 times. The pipette tip was prewet by aspirating and dispensing an aliquot of the sample solution three times.1 With the plunger depressed to the first stop, the tip was immersed approximately one millimeter into the sample solution and held there for a half second. The aliquot was aspirated from the sample solution by gently releasing the plunger, keeping the tip in the sample solution for two seconds before removal.
The aliquot was delivered by placing the pipette tip on the side of a glass vial at a 45° angle just above the meniscus and slowly depressing the plunger past the first stop to deliver the entire aliquot.
Each experiment consisted of two runs of ten data points each, using an adjustable 20 μL Eppendorf manual action air displacement pipette† set at 5 μL and Eppendorf disposable pipette tips, on the Artel PCS® Pipette Calibration System. Each experiment was performed as a comparison of one pipetting technique versus another.
The results for each pair of techniques were compared concerning trueness and precision. The measurement used for rating the precision of each technique was the coefficient of variation (%CV). Trueness was defined as the percent difference in mean delivery volumes between the two pipetting techniques.
The greatest discrepancies observed during this study were the differences between dry and prewetted pipette tips. Dry pipette tips consistently delivered significantly lower volumes than did the prewetted tips, a fact which other researchers have noted.2-6 No difficulty with precision was observed using either prewetting or no prewetting. However, differences in trueness of up to 7% between runs using dry and prewetted tips were noted while using the 20 μL pipette set at 5 μL. Additional experiments using a 250 μL pipette set at 25, 50, 100 and 250 μL consistently showed differences in the trueness of the volume pipetted of up to 2%.
Prewetting the pipette tip influenced trueness by increasing the humidity within the tip, thus minimizing evaporation of the solution. Similarly, increased ambient humidity minimized evaporation. The beneficial effect of prewetting was less significant with high ambient humidity. Ambient humidity for these runs was 50%.
Variation in the temperature of the solution being pipetted was observed to be the second largest cause of pipetting error in this study. Three sample solutions were brought to three different temperatures: 8.5, 25 and 30 °C. Solutions that were warmed to 30 °C consistently delivered lower volumes than room temperature samples. Similarly, solutions that were cooled to 8.5 °C delivered higher volumes than the ambient (25 °C) sample. The differences observed in this study were significant, ranging from 3 to 7%. Figure 1 below shows the effect of sample temperature on pipetting trueness.
Experiment showing the effect of sample temperature on the trueness of pipetting results, using a 20 µL adjustable air displacement pipette set at 5 µL. Ambient temperature 25.0 °C.
In addition to trueness problems seen with samples that were not at room temperature, there was some difficulty in obtaining good precision. Runs typically produced between 0.5% and 1.0% CV, although the CV went as high as 3.6% in some cases. As the solution was allowed to approach room temperature, the precision of the results improved. Similarly, if the pipette end or tip was warmed, even just by casual handling, differences in delivery volumes were observed. These differences and inconsistencies were smaller and less significant than those resulting from variations in solution temperature.
Reverse mode pipetting is a method of pipetting commonly used with viscous liquids. This is a technique in which the plunger is depressed past the first stop to aspirate the aliquot from the sample, and depressed only to the first stop to deliver the aliquot. Reverse mode pipetting can make obtaining accurate results (for the pipetting of solutions similar to water in terms of density and surface tension) more difficult. The study showed that a typical precision for reverse mode was 1.4% CV. Differences in volumes delivered by standard and reverse mode techniques ranged up to 5%. The reverse mode consistently delivered a higher volume than the standard method of pipetting.
Techniques among pipette users vary with background, personal preferences, and training. These differences in execution can affect the accuracy, precision and trueness of results being released from the clinical or research lab. To ensure pipetting accuracy, facilities should adopt standard operating procedures for pipetting techniques and ensure that all operators are trained to an adequate level of proficiency. By increasing the level of consistency in results obtained, the level of quality and credibility of the facility will be enhanced.
Other error causing techniques8 include:
Individually none of these factors resulted in an error greater than 2%. Cumulatively, however, two or more of these sources of error (e.g., prolonged delay and rate of plunger depression) could affect delivered volume significantly.
Component failure (e.g., a plunger seal or corroded piston), incorrect pipette tip, or incorrect installation of the tip can also affect your results.
†In an air displacement pipette, many sources of pipetting error are magnified by the ratio of “dead air” above the liquid level to the liquid volume in the tip.2-6 In conducting these experiments, a 20 μL pipette was used to deliver 5 μL, so the amount of dead air was increased, compared to pipetting 20 μL with the same pipette.
1. Zeman GH, Mathewson NS. “Necessity of prerinsing disposable polypropylene pipet tips,” Clin Chem 1974; 20(4)497-8.
2. Sternberg JC. “Sampling with air-piston pipettes – a critical study,” Clin Chem 1975; 21(7):1037.
3. Ellis KJ. “Errors inherent in the use of piston activated pipettes,” Anal Biochem 1973; 55:609-14.
4. Wenk RE, Lustgarten JA. “Technology of manually operated sampler pipettes,” Clin Chem 1973; 20(3):320-3.
5. Joyce DN, Tyler JPP. “Accuracy, precision and temperature dependence of disposable tip pipettes,” Med Lab Technol 1973; 30:331-4.
6. Lochner KH, Ballwegt, Fahrenkrog HH. “Factors influencing the measuring accuracy of piston pipettes with air interface (German),” J. Lab Med 1996; 20(7/8)430-440.
7. Ylätupa Dr. S. “Choosing a Pipetting Technique Affects the Results of Your Analysis,” European Clinical Laboratory 1996. 10:14.
8. 10 Tips To Improve Your Pipetting Technique: artel.co/resource-library/10-tips-to-improve-your-pipetting-technique/