Depending on the size of your organization and how many pipettes it uses, calibration may occur off-site through a third-party service provider, through an in-house technical team, or by end users themselves. Regardless, pipette calibration begins with a reliable analytical balance.
Gravimetric volume determinations use the well-characterized properties of water to convert weights into volumes. Specifically, temperature, atmospheric pressure, and ambient relative humidity combine to give a Z-factor that corrects for volume/weight differences according to those three properties.
“When testing pipettes, you must maintain proper humidity in the measurement area,” explains Markus Jansons, product manager at A&D Weighing (San Jose, CA). “Micropipettes deliver very small volumes. You don’t want evaporation to occur before you get your measurement.” Maintaining high humidity becomes critical with very small dispensed volumes.
Vendors sell both dedicated pipette calibration systems and add-ons that work with ordinary analytical balances. An example of a dedicated pipette test system is A&D Weighing’s AD-4212B-PT, which consists of a weighing cell (the guts of a balance without the draft shield or front panel), a control module that calculates the Z-factor, an evaporation trap, software, and an RS-232 port for data export. A&D also sells the BM-014 pipette accuracy testing kit, which is designed for use with standard, general-use microbalances.
O-ring failure is the culprit behind most out-of-spec pipettes. Over time, these seals are subjected to fumes from organic solvents, inorganic contaminants, and general use. Jansons suggests that labs invest in a leak tester that uses vacuum rather than air. “Air blowing through a pipette can introduce foreign materials,” he says. “Leak testing is something users can do every day if they like.”
Most companies outsource pipette calibration. In fact, A&D sells most of its calibration tools to dedicated calibration companies. But as Jansons notes, many labs still maintain in-house calibration services, for some as a cost-cutting strategy
Tips and tricks
An effective pipette calibration begins with a standard calibration protocol and a properly calibrated balance. Improvising the protocol is a terrible idea. Labs that lack the approved calibration fluid (usually distilled water) should not substitute, say, ten percent isopropanol.
Do-it-yourselfers should verify that their balance is calibrated and, if following ISO standards, use traceable calibration weights and good weighing practices to assure the best possible balance accuracy. Routine manufacturer or third-party servicing is the easiest way to achieve this. Similarly, users should maintain their pipette for optimal performance through regular cleaning according to the manufacturer’s recommendations.
While a good deal of leeway exists in the application of published calibration methods, generally a five-decimal-place (accurate to 0.01 mg) or better balance is all that is needed.
Weighing accuracy depends on the performance expectation for the pipette. Devices that dispense low microliters or even fractions of microliters require greater calibration accuracy than do those operating in the 50-to-100-microliter range.
“As long as you can apply the formulas and follow good operating practices for both weighing and pipetting, almost any balance can be a calibration balance,” says Andrew Hurdle, market manager for laboratory products at Ohaus (Parsippany, NJ). “Every pipette manufacturer publishes tolerances for its instruments in its instruction manuals, but these are derived under ideal conditions. So if you’re calibrating to those specific tolerances in your lab, you probably won’t achieve them.”
Hurdle advises that pipette calibration occur not under ideal environmental conditions but under real-use conditions, using the same pipette tips that are used every day. “Third-party tips may not achieve the same fill volume as the manufacturer’s own (or recommended) tip,” he says. Also, when sending pipettes out, try to provide the service organization with typical conditions in your lab.
For additional resources on analytical balances, including useful articles and a list of manufacturers, visit www.labmanager.com/balances