Pipette tips appear to be technically simple, almost commoditized, yet their design and manufacture require significant thought and engineering. “That tip you discard after each pipetting step, costing just a few cents, embodies technical sophistication that’s critical for pipetting accuracy,” says Brian Perry, PhD, senior product manager at Rainin Instrument (Oakland, CA).
Some liquid handling applications demand purpose-driven pipette tips with features like low retention or binding, used for high-viscosity liquids containing sugars or glycerin or highly concentrated salts.
Exquisite engineering notwithstanding, no pipetting system performs reliably if the tip discharges contaminants during operation. Slip agents, which are chemicals employed during the manufacture of injection-molded plastic parts to enhance manufacturability, are one potential contamination source. Tips not manufactured under pristine conditions and thoroughly inspected may harbor adventitious biological or chemical contaminants as well. For plastics, of which tips are made, leachables and extractables may affect data fidelity and experimental results in insidious ways.
“Even tips free of any detectable superficial contamination may release leachables if their materials of construction are poorly formulated,” Perry adds. Buffers, salts, acids, bases, and organic chemical constituents may initiate leaching of residual monomers, polymerization agents, or even metals.
Minimum compositional requirements should therefore include inert polymers that withstand chemical challenges, gamma- and electron beam-radiation, and autoclaving. Tip polypropylene should possess sufficient clarity to allow sample visualization, and sufficient suppleness to facilitate accurate touch-off during dispensing. With typical draw/dispense volumes in the microliter range, molding quality, physical integrity, and uniformity become critical for accurate, reproducible liquid movement within the pipette tip.
Margin of error
Jon Harkins, product manager for consumables at INTEGRA Biosciences (Zizers, Switzerland), observes that for pipette tips, fit dictates function. The company’s GripTip design, used in all its pipetting systems, mitigates tips loosening, leaking, and falling off.
GripTips incorporate a “positive stop” that causes pipette tips to load to exactly the same height. “With generic pipette tips, if you load too aggressively the tip ejects with difficulty,” Harkins says. Generic tips expand when loaded and fall off to return to their original shape.
Tip height is critical, particularly in multichannel pipettes and devices where 96 or 384 tips are expected to draw and deliver precisely the same volume. Tips seated too high on a pipette tip fitting may not reach fluid level and thereby aspirate air. Failing to make contact when touching off during dispensing causes liquid to remain within the tip. Affected wells become dead points.
These problems are magnified with today’s ultra-lowvolume pipetting. The volumetric margin of error shrinks when dispensing 0.2 microliters compared with 500 microliters. “In many cases, all you’re pulling and delivering is a drop delivered at touch-off,” Harkins observes. “If your tips are not perfectly straight and aligned at the same height, that drop will remain in the tip.”
Contamination and carryover
Contamination and cross-contamination are perennial issues in pipetting, says Karla Mantia, global product manager for pipette tips at Thermo Fisher Scientific (Pittsburgh, PA). “Pipetting creates aerosols of the pipetted sample that are suspended in the air space inside the pipette tip between the drawn liquid and the pipette nose cone.” Absent some sort of barrier, aerosols enter the pipette, remain inside, and carry over to subsequent samples. Cross-contamination occurs as previously aspirated air is dispensed along with the sample.
Many highly sensitive reactions will yield erroneous results in the presence of contaminants; for example, polymerase chain reaction (PCR). The list of PCR inhibitors is long: bile salts, urea, and calcium ions, to name a few.
Filtered pipette tips prevent aerosols from reaching the innards of the pipette and carrying over to the next liquid transfer. Several filter and barrier designs are available, constructed from different materials and each with specific effectiveness related to contamination control. “Where filtered tips keep aerosols from entering the pipette, a self-sealing barrier keeps out liquids as well, providing superior protection,” Mantia says.
Filter tips prevent contamination of the pipette and sampleto- sample cross-contamination. DNA/RNA applications often employ these tips to mitigate the activity of DNases and RNases, enzymes that hydrolyze nucleic acids.
“Customers working with viruses should also consider filter tips to avoid transferring potentially pathogenic agents from virus stocks to cultured cells,” says Kayla Hager, product manager for consumables at Eppendorf (Hauppauge, NY). Filter tips are also appropriate for manipulation of radioisotopes, which many labs still use due to legacy methods or the unavailability of appropriate fluorescent labels. Anyone who has ever worked with radionucleotides recognizes how easily surfaces—even those that seemingly do not come into contact with radioactive materials—become “hot.” Counts transferred from one experiment or process to another are result-destroyers.
Protect the pipette
In a filter-fitted tip, a barrier exists between the nose cone of the pipette and the liquid draw region of the tip. The design must allow airflow, however, or aspiration will not occur. The filter catches aerosolized particles like aqueous droplets, DNA, or virus particles. Tips are mostly made of polypropylene, but filter composition varies among manufacturers.
Filter and barrier tips cost more than their unmodified counterparts based on materials, design, and manufacturing. The increment is small, however, compared with the cost of reagents, buffers, time, and labor associated with rework due to contamination, or pipette repair or replacement. These factors alone justify the use of barrier tips.
“Sample contamination aside, one must consider protecting the pipette itself,” Mantia adds. Aerosols and liquids introduced through over-pipetting can eventually cause corrosion or compromise seals. This is more likely to occur over time if the user fails to clean and service the pipette periodically, and leads to physical deterioration and loss of accuracy.
The degree of protection depends on the filter/barrier type. Eppendorf offers standard Dualfilter T.I.P.S.® that provide aerosol protection but allow samples to draw beyond the barrier if liquid is accidently over-pipetted. The company also sells tips that seal off on contact with liquid to prevent liquid from rising above the barrier.
Customization is rare in the pipette tip world outside of packaging. Tips normally come in a rack of 96 but customers sometimes ask for packages of ten or 20, or even individually packaged tips. These are useful for clean room processes where workers bring in only the materials and equipment they need. Over the years, manufacturers of pipettes and pipette tips have improved products to make manual liquid handling easier and more ergonomic. While welcome, innovation has produced a somewhat chaotic marketplace.
An ISO standard, 8655-2 recommends that customers should use pipettes and tips from the same manufacturer, even for “open system” products. Hager explains that no industry standards exist for dimensions of nose cones or tips drawing specific volumes. “But when you purchase a pipette from a manufacturer that also sells the tips, the pipette is calibrated for that specific tip. Using another supplier’s tips requires recalibration.”
For additional resources on pipettes, including useful articles and a list of manufacturers, visit www.labmanager.com/pipettes