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From Weight to Volume

Gravimetric solutions for pipette calibration

Isabelle Schrepfer (Mettler-Toledo GmbH, LHCD (Liquid Handling Calibra­tion Devices))

Regular testing is essential to ensure that pipettes work accurately and precisely. However, measuring the volume of liquids is subject to inherent risks, particularly when the volume is small. Gravimetric solutions for testing and calibrating pipettes offer precise and regulatory compliant solutions to ensure that these critical lab instruments always perform within specifications.

Widely used in biology and chemistry applications, pipettes play a key role in the laboratory. In projects, where the product development process is long and a great deal of money has been invested, it is important that the impact of the pipette’s accuracy on the quality assurance process is considered.

The need for regular testing

Each pipette is unique and demonstrates its own measurement uncertainty. Testing and calibration of pipettes is essential to ensure the dispensed volume is within the specified process tolerances. Only with regular testing, can companies be sure that their pipettes remain within tolerance, and only with rigorous documentation can they be sure of fully traceable results and satisfy their auditors. Without regular testing, it may be impossible to determine if and when a pipette has gone out of tolerance, which may result in very costly reworking and a lot of wasted time.

Determining volume accurately by weighing

There is no independent standard reference volume. The liter is officially defined as a cubic decimeter and
so is based on length rather than volume. The volume of a liquid is dependent on temperature and pressure which makes it extremely difficult to define and manage a standard based directly on volume. By contrast,
a weight is not affected by these influences, although weighing equipment must be calibrated to the installed environment. Testing pipettes is also complicated because the dispensed volumes may be as small as 1 µl. This is not only very difficult to measure but evaporation of the test water has a significant influence on the measured result. An indirect method of determining the volume is therefore necessary.

The traditional solution for determining volume is gravimetrically. Many regulations are in place to
stand­ardize gravimetric determination. All the tests and adjustments for a correctly managed balance can
be traced back to the international prototype kilogram. Regular testing of correctly installed balances ensures compensation for environmental conditions such as temperature and atmospheric pressure. These influences do not affect the weight of the test water, but may change its volume. The measurement uncertainty of a balance can also be determined by standard procedures so a volume can be calculated with a known degree of confidence.

Pipette calibration

ISO 8655 not only describes the construction requirements for piston operated pipettes but includes testing pipettes by the gravimetric method to determine the measurement uncertainty, and specifies the balance resolution required for specific volumes. The test water from the pipette is dispensed directly into the receptacle on the balance. The volume of the liquid is calculated by applying a correction factor to the weight which is directly related to the density of the test water and allows compensation for temperature and atmospheric pressure. Typically, a series of ten measurements are taken to provide an average. The standard deviation, or spread, of the results is a measure of the repeatability or uncertainty of the pipette.

Calibrating micropipettes

To determine volumes as small as 1 µl gravimetrically, the ISO standard specifies that a microbalance is necessary in order to meet the required tolerance specifications. METTLER TOLEDO’s XP26PC is a microbalance specifically designed for the calibration of micropipettes and is suitable for determining the weight of such small volumes. The integrated evaporation trap minimizes evaporation from the dispensed test water to ensure a more accurate result and decrease stabilization time. The cover to the evaporation trap opens automatically when the pipette breaks the integrated light beam. This creates an ergonomic handling process and minimizes operator fatigue during this highly repetitive process.

Converting the weight to volume

Due to the number of pipette tests which must be carried out, the complex conversion process is best performed with a dedicated solution. METTLER TOLEDO’s Calibry Pipette Calibration software supports the entire calibration process with full user guidance and comprehensive pipette management functionalities. Calibry ensures compliance with ISO 8655 and not only automatically converts the weight into a volume but also translates weight uncertainty into volume uncertainty. By saving all calibration data in a secure database, Calibry also assists users to fulfill FDA 21 CFR Part 11. The full calibration history of individual pipettes is easily accessed and can be printed in a report at any time.

The latest release of the Caliby software is now also compatible with RFID-enabled pipettes, such as Rainin’s Pipet-Lite XLS series. Calibry can read and write information on the RFID tags such as the pipette’s serial number, its assigned project and recalibration date. This presents a major advance in the monitoring of pipette calibration dates and provides companies with an increased level of security in their day-to-day processes.

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