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
- Temperature measured 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.
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
• There are no consistent standards for how manufacturers set their
• 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.