Forward and reverse mode Use of the appropriate pipetting mode has
one of the biggest influences on the accuracy of the volume delivery. In forward mode, the plunger is depressed to the first stop, the pipet tip is then immersed in the sample solution, and the sample is subsequently aspirated. During delivery, the plunger is depressed beyond the first stop (blow-out stop), forcing all the liquid out of the tip. Standard procedure for pipet calibration prescribes using this forward mode and aqueous sample solutions.3,5
In reverse mode, the plunger is depressed be- yond the first stop (to the second stop) before immersing the tip in the sample, aspirating more than the desired sample volume. The desired volume is delivered by depressing the plunger to the first stop, retaining the additional sample in the tip. While this pipetting mode is recommended for use with viscous or volatile solutions, using reverse mode with aqueous solutions leads to significant overdelivery of up to 2.3% RI and contributes up to 0.7% CV.
Consistent plunger speed
and pressure Depressing and releasing the plunger with con- sistent speed during aspiration and dispensing of the liquid aliquot is important for achieving precise and accurate results. The type of pipet, tip, and sample solution will determine the op- timum pressure needed to move the plunger with a consistent and appropriate speed. Our studies indicate that a slow aspiration speed may result in underdelivery of up to –1.1% RI and contribute up to 0.7% CV.
Position of tip during aspirating
and dispensing Holding the pipet in a vertical orientation and preventing the tip from touching the side or bottom of the sample vessel will ensure an optimal and undisturbed hydrodynamic flow of the sample during aspiration. Further, it is important not to drag the tip along the wall of the source vessel after aspiration, because this may lead up to –0.7% RI and 0.6% CV.
When dispensing the sample, it is recom- mended to touch the pipet tip against the side of the receptacle, while the pipet may be held at a 45° angle. With the exception of pipetting
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany AMERICAN LABORATORY • 15 • JUNE/JULY 2014
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very small volumes, it is not recommended to immerse the tip into already present solution in the receiving vessel, because this may lead to overdelivery if droplets are clinging to the outside of the tip, and significantly increases the risk of cross-contamination.
Pause after aspirating Once the aliquot of sample solution has been aspirated into the pipet tip, it is important to
pause for about 1 sec with the tip still immersed in the source liquid, allowing the sample to “settle” in the tip. Removing the pipet tip prior to allowing the vibrational motion of the liquid to settle will introduce errors in the precision and accuracy, up to –0.6% RI and 0.4% CV in our studies. Allowing the tip to remain in the liquid for too long, however, will result in significant underdelivery, up to –2.3% RI and 1.6% CV. The magnitude of these errors depends on the
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