In practiced hands, a properly calibrated pipette is an accurate and precise instrument. As with any precision instrument, however, improper technique can negatively affect test results. A guide to successful pipetting is given below.
Operator training
Pipetting proficiency is one of the most critical elements to ensure accuracy and precision, particularly in the face of high personnel turnover, growing laboratory workloads and increasingly complex tests. Thorough training regimens should be documented and include regular proficiency assessments, even if this is not a regulatory requirement. Continually employing consistent pipetting technique across the entire laboratory workforce ensures consistency of test results.
Pipette calibration
Like all mechanical instruments, pipettes are subject to random failure. Electronic and mechanical pipettes rely on internal high-precision components to function properly. Silent mechanical failures, which are often not visible or evident by the pipette’s “feel,” can be detrimental to critical diagnostic tests and experiments.
Systematic calibration programs and interim verification checks must be implemented to offset risk from out-of-tolerance pipettes and to quickly identify those that are failing. The specified intervals for routine calibration depend upon how pipettes are stored and used, the criticality of their application, the laboratory’s risk tolerance and, in some cases, the regulations in effect for that laboratory. The longer a defective pipette remains in service without a performance check, the greater its possible liability. Frequent checks (calibration or verification) to identify malfunctioning pipettes will reduce the risk to the lab.
Sample preparation
Sample preparation for most analytical and production processes requires accurate, precise liquid handling. Whether an accurately weighed solid sample is dissolved or liquid samples are diluted, the amount of solvents used in these steps is crucial as they determine the resulting concentration of the materials of interest.
Inaccurately prepared samples can lead to just as many errors in final test results as inaccurate dispenses further downstream in the process. It is therefore imperative to use properly calibrated liquid-handling instruments at all stages of an analytical or production process.
It is advisable to use the tips recommended by the pipette manufacturer. Every pipette shaft has a specific taper angle that is designed to fit optimally to the recommended tips. Tips have to form a tight seal with the pipette shaft, as a leaky seal contributes to errors in pipetted volumes. When using tips from other than the pipette’s manufacturer, the user must carefully check the fit and confirm there is no gap at either the top or bottom of the shaft, and that the tip is properly aligned and fits snugly.
Using a pipette and tip combination that corresponds closely to the volume to be pipetted will yield better results than attempting to pipette a very small volume with a very large pipette and tip .
Laboratory environment
Temperature and relative humidity are usually programmed to provide a comfortable working environment in the laboratory. Instruments also have a comfort zone. Air displacement pipettes use an air cushion to couple the plunger to the aspirated liquid in the tip. This air cushion, or captive air volume, closely follows the ideal gas law and is therefore susceptible to changes in temperature and pressure.
For accurate and consistent pipetting results, the sample solution, pipette and pipette tip should be equilibrated to the same temperature. When pipetting samples that are warmer or colder than the pipette and tip, the air cushion can expand or contract, resulting in less or more aspirated sample, respectively.
Relative humidity in the laboratory impacts sample evaporation, not only due to uncovered receptacles on the lab bench, but also within the pipette tip. To reduce the amount of evaporation, a pipette tip should be prewetted several times by aspirating the sample and dispensing it back into the sample vessel. This increases the humidity inside the pipette tip, reducing the amount of sample evaporation and thereby the accuracy of the pipetted volume. The lower the relative humidity, the more prewetting cycles should be used; three to five cycles are usually sufficient.
When temperature equilibrium is not possible (e.g., when pipetting solutions at 2–4 °C or 37–40 °C), the set volume of the pipette may need to be adjusted, or positive-displacement pipettes may need to be used.
Pipetting modes
Two pipetting techniques are used in the lab: forward mode (also known as standard mode) and reverse mode. For pipetting most aqueous solutions, forward mode should be used. It typically yields better accuracy and precision than reverse mode and is used by manufacturers to calibrate their pipettes. Reverse mode is recommended for viscous solutions, because forward mode for this application can result in underdelivery.
To pipette with forward mode, the plunger is pressed to the first stop and the tip is immersed in the sample. The plunger is then released to aspirate the aliquot. Finally, the entire contents are dispensed by pressing the plunger to the second stop, the blow-out step.
To engage reverse mode, the plunger is depressed to the second stop before immersing in the sample, which effectively overaspirates it. It is then depressed only to the first stop to deliver sample, leaving some in the tip.
Aspirating
Figure 1 – a) During aspiration, the pipette is held in the vertical position and the tip is immersed to the proper depth (note: the user should avoid touching the side walls or bottom of the sample container). b) Sample is dispensed with the tip touching the receptacle wall at an angle and dragging it slightly along the wall.Pipetting should be slow and steady. Operators should pause immediately after aspirating, with the pipette tip still in the sample liquid. Failure to do so can lead to inconsistent aspiration volumes because the liquid requires about a second to settle after aspiration is complete. A pause of about one second is acceptable, but anything longer than that may result in underdelivery.
It is also important to press and release the pipette’s plunger with a consistent, moderately fast motion. Moving the plunger too fast or too slow will negatively affect the aspirated volume. Inconsistent speed will decrease precision of the pipetted aliquots.
Holding the pipette perpendicular to the liquid surface and avoiding touching the inside walls of the sample container will minimize errors due to surface tension or partially obstructed flow (see Figure 1a).
Dispensing
Consistency and care are critical also when dispensing sample. Before dispensing, pipettes should be checked for droplets on the outside of the tip, with visible droplets removed very carefully with a lint-free cloth. This should only be done if absolutely necessary, however. Liquid can be wicked from the tip opening, causing sample loss and underdelivery.
Once clean, the tip should be placed against the receptacle wall and the plunger pressed to the second stop when using forward mode. The tip should then be slightly dragged up the receptacle wall to allow all liquid to be drawn from the tip (see Figure 1b). Repeated actions produce repeatable results—the plunger should always be pressed and released with consistent speed and pressure.
Tip immersion
Tip immersion depth is important and requires subtlety: if it is too shallow, there is a risk that air can be aspirated; too deep in the sample, and droplets may collect on the outside of the tip and more sample might be forced inside.
Pipetted volume will determine the correct immersion depth,1 generally 2–4 mm below the surface for volumes from 1 μL to 1000 μL, and 3–6 mm for volumes larger than 1 mL. The pipette should be held in a vertical position (tilting it may cause a change in sample volume) and the tip should not be allowed to touch the sides or bottom of the sample vessel.
Avoid heat transfer from hands
The barrel of a pipette will heat up after prolonged handling due to heat transfer from the palm of the hand. This can lead to an expansion of the captive air volume and ultimately decrease the delivered sample volume. To mitigate this, the pipette should be held loosely and returned to its stand or set down between deliveries.
Conclusion
Pipetting requires concentration and precision. With proper training, preparation and technique, and by working with regularly calibrated pipettes, operators can consistently produce accurate and reproducible results.
Reference
- ASTM E1154-14, Standard Specification for Piston or Plunger Operated Volumetric Apparatus; http://www.astm.org/DATABASE.CART/HISTORICAL/E1154-89R03.htm
A. Bjoern Carle, Ph.D., is a product manager at Artel, 25 Bradley Dr., Westbrook, Maine 04092-2013, U.S.A.; tel.: 888-406-3463; e-mail: [email protected]; www.artel-usa.com