Pipette Calibration advice (2023)

Pipette Calibration advice (1)

How to Calibrate a Pipette
This is often a misunderstood element of the pipette service and maintenance regime as pipettes are calibrated against a standard and according to strict protocols. The actual pipetting that happens in a laboratory (subsequent to a calibration and service) on a true sample may well produce different results and causes ambiguity and concern.

For example a pipette that has just been calibrated in a temperature and humidity controlled environment by trained operators using the manufacturer’s tips may see different results if the pipette is then used in a laboratory. Especially if the laboratory is in a hot country, using a viscous serum sample taken straight from the fridge using poorly fitting tips and with a poor technique. This is obviously an extreme example but one or more of these elements can often be found in normal laboratories. There is no “normal” laboratory however and therefore any specific requirements for calibration should be notified to the calibration company (fluids used, non-manufacturers tips etc) or otherwise standard approved protocols will be followed by these companies.

Calibration is predominantly undertaken by gravimetric analysis and is the method most commonly used by laboratories accredited to ISO17025. There are colorimetric methods in use and these are often used for checking pipettes and for non-accredited calibration. Gravimetric analysis is preferred due to the simplicity and the traceability to an absolute standard. Gravimetric methods are also often recognsied as a more economical way of calibration.

Gravimetric analysis for pipette calibration entails dispensing samples of distilled water into a receiving vessel in a precision analytical balance. The density of water is a known constant, the temperature, barometric pressure and humidity are recorded (the Z-factor used in the final mass calculation) and kept within certain limits and thus the mass of the dispensed sample provides an accurate indication of the volume dispensed.

All the elements of this process are themselves audited to ensure compliance and chain of custody for accurate measurements. Hence the micro-balances, the thermometers, chart recorders; barometric devices, software and users are audited by external bodies to ensure accuracy at all levels of operation. Use of controlled conditions increases the likelihood of any calibration being more reliable and “Calibration Rooms” with the correct atmospheric conditions and with vibration free surfaces will give more reliable results and are compulsory for ISO17025 requirements with fine tolerance uncertainty budgets.

As an example of environmental conditions and equipment the following would be typical of an accredited laboratory

- Temperaturemeasured to ± 0.01 degree
- Temperature controlled to within 0.2 degree C
- Humidity is measured to ± 2%.
- Barometric pressure is measured to 0.01 mm Hg.

Pipette Calibration advice (2)

In-House Pipette Calibration

Many institutes calibrate and service pipettes themselves. This requires the correct equipment and should be undertaken in monitored and recorded conditions by experienced individuals. Balances are freely available to purchase specifically for this purpose with “evaporation traps” and software can be purchased from a number of companies to assist in this. This software should ideally have inventory control and training/monitoring options for staff to ensure continued good techniques.

Most manufactures provide tools and instructions for calibration. The only issues arise when there is a need for accredited results and certificates and/or the pipettes cannot be re-calibrated within specification (the manufacturers publish these). The pipette may therefore require maintenance or repair. The pipette may be repaired in-house with spares purchased from the manufacturer or a third party or sent off for closer inspection. The majority of air-displacement manual pipettes can be fixed and re-calibrated by trained technicians or engineers; however electronic pipettes may be more complex and require intervention of an experienced company.

Laboratories and operators need to be aware that applying pipette manufacturers' specifications as the tolerance limits for in-lab testing may prove difficult to achieve. The performance attained in the operator’s laboratory may not reflect the published credentials from the manufacturer. The reasons for this are as follows:

• A pipette’s performance as mentioned previously is influenced by environmental factors such as temperature and humidity.

• The performance can be affected by the test fluid's temperature and viscosity.

• There are no consistent standards for how manufacturers set their performance claims.

• The skill of the pipette operator plays a very important role in the precision and accuracy of the pipette.

• The type of pipette tip used in the testing also affects results. The manufacturers tip is always recommended, but if the operator commonly uses another tip then this should be the tip of choice.

• The environmental conditions do need recording at all times and incorporating in to the analysis of performance.

• Statistical factors may be a problem caused by the number of data points and erroneous sporadic pipetting techniques, plus calculation methods may all impact the results.

The tolerance limits achievable in an operators laboratory need to be addressed prior to documenting an in-house calibration regime.


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