All of the air-displacement manual pipettes (90% of the pipette market) use the same principles for measuring and transferring accurate amounts of fluid. They operate by use of a piston mechanism which is activated by either the thumb or hand and which subsequently generates a vacuum by vertical travel of the metal or ceramic piston within an airtight shaft. As the piston moves up, driven by the release of a plunger, a vacuum is created in the space left by the piston. This vacuum is then replaced by air. When a disposable tip is attached to the tip cone and immersed in liquid, the liquid is then drawn into the tip and subsequently dispensed by depression of the plunger.
Factors Affecting a Pipettes Performance
A pipette’s design and use is fundamental to the accuracy of a pipette and its continued precision.
An operator should be trained in the correct use of a pipette and occasionally compared against standards to ensure continued good practise.
Most users will be aware (if made conscious of the fact) that both air temperature and relative humidity can affect a pipettes performance as can the temperature of the sample (freshly thawed serum for example).
In early pipettes heat transference from operator's hands caused small changes in the pipetting volumes. The heat was absorbed through the handle of the instrument and transferred to the components inside, the interaction between which caused these discrepancies. More modern pipettes have air spaces and less thermo conductive materials and the “heat Effect” is considered minimal. If this is a concern however the best technique for maximum accuracy is to employ multiple pipettes and rotate them often in use.
One of the most common causes of inaccuracy and mistakes is operator fatigue. Even the best trained and experienced operator will see a decrease in accuracy and precision as time increases. Pipette calibration companies restrict the number of pipettes a technician can calibrate in a single day for this reason. In the last 20 years this effect has also become recognised by Health & Safety due to increasing numbers of Work Related Upper Limb Disorders (WURLD) often known as Repetitive Strain Injury an example of which is Carpal Tunnel Syndrome. Many pipettes have now reduced thumb forces needed to use a pipette and have decreased the weight of pipettes to ease a users discomfort when pipetting for long periods. Another solution is choosing an electronic pipette which significantly reduces hand fatigue. Once the operating button is touched the pipette always operates always the same way producing user independent accuracy and precision.
Component integrity is critical to the performance of a pipette. With any mechanical device where several components interact in movement there is friction, “wear and tear”, fractures, weakness and loss of tolerance. This is no different in a pipette where the objective is to create a precise vacuum through use of a piston, mechanical travel, springs, and rubber and/or polymer seals. Add to this the possible corrosive effects of fluid, poor pipette handling, damage to external components, volume adjustment problems and you can see the reasons for checking performance and calibration.
Maintaining Accuracy & Precision
For maintaining accuracy and precision even through repeated pipetting regimes, pipettes should be calibrated at periodic intervals. The interval at which a pipette needs calibrating depends on several factors:
- The skill and training of the operators and the care with which the pipettes are used.
- How intensively the pipettes are used.
- The type of liquid dispensed by the pipettes. Volatile liquids or corrosive liquids may emit vapours or come into contact with metal pistons, springs, seals and o-rings.
- The accuracy and precision required by the pipette. Pipette applications that require superior accuracy also demand more frequent calibration.
- There is not absolute for calibration intervals but each institute or organisation should produce their own risk analysis/calibration requirement document. A pharmaceutical company manufacturing drugs or a underataking a clinical analysis of specimens will have more stringent requirements than a school chemistry class for example.
- Under “normal” conditions, most pipettes can be calibrated semi-annually (every six months) and provide satisfactory performance. Institutions that are regulated by the FDA or GMP/GLP regulations will benefit from quarterly calibration. Very critical applications may require monthly calibration. May laboratories will undertake weekly “checks” on a balance as well, but even this needs doing carefully. Clearly these are general guidelines.
- Many pipette calibration companies will offer varying levels of service/calibration, depending on the operators/institutes requirements, for example.
1. A basic calibration often used by teaching & university labs with broad specs.
2. As above but supplied with a basic calibration certificate.
3. Regulated laboratories which require data point results with pass/fail status according to the manufacturers or operators requirements.
4. An ISO/IEC 17025:2005 accredited service used by laboratories who want ISO 17025 accredited pipette calibrations and require only with full certification, and statistical analysis with pass/fail according to specified criteria.
Even these levels of calibration which are offered can differ substantially due to the number of data points used, the “as-found” and “as-left” requirements and manufacturer’s specifications/users requirements. This can become even more complex when multichannel pipette calibration is required or repeat pipette measurements on electronic or stepper pipettes.
It may be useful to open a dialogue with the service companies at the more extreme levels as even the certification may need to be tailored to a laboratory's needs.