Pipetting microliter volumes of liquid has become one of the most frequently performed laboratory procedures. The speed, convenience and cost benefits of microliter‑volume, air‑displacement pipettes drive the frequent use of these precision instruments. However, pipettes, like all mechanical instruments, are subject to failure, and this, along with the technique of operators, can have a significant impact on experiment accuracy and precision. As a result, it is more important than ever to ensure the accurate and consistent operation of pipettes, especially at the small microliter or nanoliter volumes typical of today’s analytical applications.
Laboratories have traditionally used gravimetric calibration or outsourced calibration services to ensure against pipetting error. However, these methods have several drawbacks that create impediments. With Artel’s PCS® Pipette Calibration System, a robust technology based on Ratiometric Photometry, laboratories can quickly and conveniently verify pipette accuracy and precision, and can cost effectively ensure pipette performance throughout the laboratory year, and not just at predetermined calibration cycle times.
This article will present data showing the frequency and magnitude of pipetting error in laboratories, and how the PCS can be used to reduce these errors and strengthen data integrity.
Air‑displacement pipettes are complex precision instruments. The accuracy and precision of a properly functioning pipette in the hand of a trained operator are exceptional, even for volumes as low as 5 microliters. Unfortunately, in day-to-day operations, pipettes fail due to wear and tear, unexpected mishaps and operator technique variation. These sources of error impact liquid delivery volumes and, consequently, data integrity. To demonstrate the origin and prevalence of pipetting error in laboratories, three data sets were collected. First, baseline data were collected using trained operators and a properly functioning pipette. Next, pipette malfunction was evaluated, and lastly, operator technique errors were measured.
To establish baseline conditions, two trained pipette operators used a properly functioning 20-microliter pipette set at 5 microliters to pipette 10 sets of five deliveries. The pipettings were done under laboratory conditions at normal speed (see Table 1). In all cases, the accuracy was better than 2% and the precision better than 1%.
Accuracy and Precision Results from Trained Operators using Properly Functioning Pipette
To test pipette failure rates, the same trained operators evaluated the performance of pipettes in use in four laboratories at an internationally recognized biomedical research institute. Fifty-four 20-microliter variable‑volume pipettes were tested at 5 microliters using the PCS, and the inaccuracy and imprecision of each pipette were plotted (see Figure 1).
The data showed that the ordinary laboratory pipettes were not in good condition. Only 12 of the 54 pipettes (22%) produced accuracy and precision results as good as the baseline case, and five pipettes (9%) were severely underperforming, with inaccuracy worse than 20%. These data show that the mechanical condition of laboratory pipettes cannot be taken for granted.
Next, operator skill was tested. Fifty-four technicians working in QC laboratories at four pharmaceutical companies were tested. Each used the same properly functioning 20-microliter pipette set at 5 microliters. Their technique was tested with the PCS under actual laboratory conditions. The percent inaccuracy and imprecision data are shown in Figure 2. Only 19% of the laboratory personnel produced results as good as or better than the trained operators in the baseline case. Six operators (11%) produced errors more than 10 times larger than the baseline case.
Properly maintained pipettes in the hands of skilled operators are capable of reliably delivering 5 microliters of liquid with better than 2% inaccuracy and 1% imprecision. However, few experienced pipette operators can meet this standard without some training in pipette technique. Moreover, many pipettes in working laboratories are malfunctioning and in need of repair. The errors contributed by pipette failure and untrained operators can be very large, and are frequently more than 10 times the optimal level.
Errors of this magnitude, especially in pipetting, one of the most frequently performed laboratory tasks, are unacceptable. Clearly, diligent maintenance of pipettes and training of operators would increase overall laboratory accuracy and efficiency. Yet, few laboratories have attained this optimal state of pipetting proficiency.
One explanation for this failure is the limitations of the gravimetric pipette testing method when working with microliter volumes. The gravimetric method, which weighs liquids on balances to determine volume, was developed when protocols were at the milliliter scale or higher. At the microliter level, the gravimetric method is time consuming, technically challenging and subject to human error and variation caused by environmental conditions. Outsourced pipette calibration services can repair failed pipettes but do not address the issue of operator training.
Faster and more accurate than gravimetry, photometry is becoming the method of choice for pipette calibration. Measuring light absorption to verify volume, photometric pipette calibration systems are generally five to seven times faster than gravimetric methods and have greater accuracy for volumes less than or equal to 200 microliters. This makes in‑house pipette checking and operator training significantly more practical.
PCS is a rapid, easy‑to‑use photometric instrument and reagent system unique for its dual dye Ratiometric Photometry approach resulting in improved accuracy and precision. The system includes a reagent kit and a photometric instrument (Figure 3). The instrument guides the operator through the test plan for the selected pipette and an optional pipette tracking software program is available to simplify scheduling and data management.
Speed, ease of use and accuracy make the PCS well-suited for training technicians in proper technique as well as for frequently checking to detect pipette failure. PCS provides an opportunity for all laboratories to increase accuracy and efficiency by controlling pipetting error and thereby strengthening data integrity.
Richard Curtis, PhD, is Chairman and Chief Technology Officer at Artel and shares executive company management responsibilities. Dr. Curtis also manages Research, Development and Engineering activities and directs the advancement of Artel’s core technology, new product concepts, new generations of current products, manufacturing engineering and quality assurance. He has been responsible for the acquisition of patents in photometric analytical systems, electronic circuitry, optics, and engineering physics. Dr. Curtis earned a BA cum laude in Physics at Harvard and a PhD in Nuclear Physics at Brown University.